CN106987571A - A kind of Cas9 nucleases F916P and application thereof - Google Patents

A kind of Cas9 nucleases F916P and application thereof Download PDF

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CN106987571A
CN106987571A CN201710344461.4A CN201710344461A CN106987571A CN 106987571 A CN106987571 A CN 106987571A CN 201710344461 A CN201710344461 A CN 201710344461A CN 106987571 A CN106987571 A CN 106987571A
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吴强
李金环
寿佳
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Shanghai Jiaotong University
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Abstract

The invention belongs to biological technical field, and in particular to a kind of Cas9 nucleases and purposes.The Cas9 nucleases of the present invention（Cas9‑F916P）, with Cas9 nucleases, it is adaptable to CRISPR/Cas9 systems, the Cas9 nucleases（Cas9‑F916P）It is that the 916th phenylalanine of wild type Cas9 nucleases the is mutated into proline to obtain.Using the Cas9 nucleases（Cas9‑F916P）Prominent broken ends can be produced by carrying out cutting to DNA double chain, and the base complementary with prominent broken ends can be added in the way of filling-in is connected, the accurate editor to the ad-hoc location of genomic DNA fragment can be realized.

Description

A kind of Cas9 nucleases F916P and application thereof
Technical field
The invention belongs to biological technical field, and in particular to a kind of Cas9 nucleases F916P and purposes.
Background technology
Since the Human Genome Project (Human Genome Project) and DNA element encyclopedia The completion of (Encyclopedia of DNA Elements) project, scientists are analyzed and identified in substantial amounts of genome Gene and DNA controlling elements [1,2].The DNA controlling elements played an important role in gene expression regulation include promoter, enhancing Son, silencer and insulator etc..However, checking that the function of most controlling elements is not tested and illustrating [2-8].Visit The function of sok gene and DNA controlling elements, can be studied by science of heredity DNA fragmentation editor.
Gene editing and the gene function modification of early stage is to realize [9-14] by gene transposition and transgenosis.With survey The development reverse genetics of sequence technology is applied to that genome is carried out specifically to be mutated [15,16].Particularly dependent on homologous The gene targeted mice of restructuring is promptly applied in scientific research [15,17,18].In addition, in mouse and zebra fish DNA The reversion and repetition of fragment are applied to study specific genome structure change [19-24].
In recent years, the short palindrome in II type Regularity interval for coming from bacterium and ancient bacterium repeats system [Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated Nuclease 9 (Cas9), CRISPR/Cas9] it is emerging genome editing technique [25-27], because it designs simple and operation It is convenient, promptly it is applied to eukaryotic gene groups editor.We utilize CRISPR/Cas9 systems real in human cell line and mouse DNA fragmentation heredity editor is showed and (has deleted, inverts and repeat) [28].Two are carried out in genome by Cas9 and two sgRNAs It can realize that the deletion of DNA fragmentation, reversion (are fallen under the repair system effect that the albumen such as CtIP are participated in after individual site targeted disruption Position), repeat, transposition and insertion (provided that donor) etc. [29-32].Pass through the genetic manipulation to DNA fragmentation editor, Neng Gouyong Come the gene expression regulation for studying protocadherin and globin and three-dimensional genome structure [28,31-33].
The editor of DNA fragmentation can be realized by CRISPR/Cas9 systems at present, but for furtheing investigate specific DNA The accurate function of section, the Cas9 nucleases for effectively realizing the accurate heredity editor of DNA fragmentation are also to be found.
The content of the invention
In order to overcome the problems of in the prior art, it is an object of the invention to provide a kind of Cas9 nucleases and use On the way.
To achieve these goals and other related purposes, the present invention is adopted the following technical scheme that：
The first aspect of the present invention is there is provided a kind of Cas9 nucleases (Cas9-F916P), with Cas9 nucleases, fits For CRISPR/Cas9 systems, the Cas9 nucleases (Cas9-F916P) are by the ammonia of wild type Cas9 nucleases the 916th Base acid is mutated into Pro by Phe and obtained.Also it is that the 916th phenylalanine of wild type Cas9 nucleases is mutated into proline to obtain .
Preferably, compared with wild type Cas9 nucleases, the Cas9 nucleases (Cas9-F916P) are to target gene group The protrusion broken ends and the ratio of blunt broken ends produced when DNA fragmentation is cut are different.
Preferably, the wild type Cas9 nucleases are SpCas9.
Further, the amino acid sequence of the wild type Cas9 nucleases is as shown in SEQ ID NO.7.
Preferably, the Cas9 nucleases (Cas9-F916P) contain the amino acid sequence as shown in SEQ ID NO.9.
Preferably, the amino acid sequence of the Cas9 nucleases (Cas9-F916P) is as shown in SEQ ID NO.9.
The second aspect of the present invention is there is provided a kind of polynucleotides, and it encodes the Cas9 nucleases (Cas9-F916P).
The third aspect of the present invention is there is provided a kind of expression vector, and it contains foregoing polynucleotides.
The fourth aspect of the present invention is there is provided a kind of host cell, and it is converted by foregoing expression vectors.
There is provided the method that one kind prepares the Cas9 nucleases (Cas9-F916P), including step for the fifth aspect of the present invention Suddenly：The expression vector containing Cas9 nucleases (Cas9-F916P) coded polynucleotide is built, then turns the expression vector Change the induced expression into host cell, separation obtains described Cas9 nucleases (Cas9-F916P) from expression product.
The sixth aspect of the present invention is there is provided foregoing Cas9 nucleases (Cas9-F916P) or its coded polynucleotide or contains The expression vector of the coded polynucleotide is used for genomic DNA fragment editor or for preparing genomic DNA fragment editor's work The purposes of tool.
Preferably, the editor includes unit point editor and many sites editor.The edit bit points of many site editors For two and more than.
Preferably, the mode of the editor includes mutation, deletes, inverts or inversion, repetition, transposition or insertion.
There is provided a kind of genomic DNA fragment edit tool, the genomic DNA fragment editor for the seventh aspect of the present invention Instrument is CRISPR/Cas9 systems, and the CRISPR/Cas9 systems include foregoing Cas9 nucleases (Cas9-F916P) or its volume Code polynucleotides or the expression vector containing the coded polynucleotide.
Preferably, the CRISPR/Cas9 systems include foregoing Cas9-F916P and one for target DNA fragment or Multiple sgRNA.It is the multiple refer to two and more than.
The eighth aspect of the present invention is there is provided a kind of genomic DNA fragment edit methods, using foregoing Cas9 nucleases (Cas9-F916P) and matched one or more sgRNA, using CRISPR/Cas9 systems to genome to be edited DNA fragmentation enters edlin.
Preferably, the editor includes unit point editor and many sites editor.The edit bit points of many site editors For two and more than.
Preferably, the mode of the editor includes mutation, deletes, inverts or inversion, repetition, transposition or insertion.
Preferably, will the expression vector and therewith containing foregoing Cas9 nucleases (Cas9-F916P) coded polynucleotide The one or more sgRNA coordinated are together transferred in cell, and edlin is entered to genomic DNA fragment to be edited.
The ninth aspect of the present invention utilizes CRISPR/Cas9 there is provided a kind of genomic DNA fragment unit point edit methods System, carries out cutting to DNA double chain using Cas9 nucleases (Cas9-F916P) as claimed in claim 1 and produces prominent fracture End, by cell itself repair system, adds the base complementary with prominent broken ends in the way of filling-in is connected.
The feature of base mutation when the genomic DNA fragment unit point edit methods can change unit point editor.
Compared with prior art, the present invention has the advantages that：
The Cas9 nucleases (Cas9-F916P) of the present invention, it is adaptable to CRISPR/Cas9 systems, the Cas9 nucleases (Cas9-F916P) containing the amino acid sequence as shown in SEQ ID NO.9, the Cas9 nucleases (Cas9-F916P) and open country Raw type Cas9 nucleases are compared, the protrusion broken ends produced when being cut to target gene group DNA fragmentation and blunt fracture end The ratio at end is different relative to wild type Cas9 nucleases.DNA double chain is entered using the Cas9 nucleases (Cas9-F916P) Row cutting can produce prominent broken ends, can be added by way of cell itself repair system is connected with filling-in and prominent fracture The base of termini-complementary, can realize the accurate editor that particular bases are added to the ad-hoc location of genomic DNA fragment.
Brief description of the drawings
Figure 1A：Cas9 carries out cutting to DNA double chain under two sgRNAs mediations and produces four broken ends, these fractures End produces DNA fragmentation in the presence of cell repair system and deletes, inverts and repeat.
Figure 1B：Situation is deleted, inverts and repeats for the DNA fragmentation in HS51 sites.
Fig. 1 C：DNA fragmentation deletes the addition that joint has " G ".
Fig. 1 D：DNA fragmentation repeats the addition that joint has " T ".
Fig. 1 E：There is the addition of " A ", " G " and " AG " in DNA fragmentation downstream reversion joint.
Fig. 1 F：For the sgRNAs of the two particular sequences, Cas9 cutting mode ratio characteristics.
Fig. 2A：Cas9 nuclease structural representations.
Fig. 2 B：β-globin RE2 sites carry out two sgRNAs of DNA fragmentation editor schematic diagram.
Fig. 2 C：Mediation of each Cas9 nucleases in sgRNA1 is counted by detecting that DNA fragmentation repeats joint connection Under various incision tips produced when being cut to genomic DNA fragment accounting.
Fig. 2 D：For upstream sgRNA1, cutting situation of the Cas9 and Cas9 mutant to target DNA fragment.
Fig. 2 E：Mediation of each Cas9 nucleases in sgRNA2 is counted by detecting that DNA fragmentation deletes joint connection Under various incision tips produced when being cut to genomic DNA fragment accounting.
Fig. 2 F：For downstream sgRNA2, cutting situation of the Cas9 and Cas9 mutant to target DNA fragment.
Fig. 2 G：Cas9 and Cas9 mutant inverts reality and the prediction ratio that side joint base is added in DNA fragmentation Example.
Fig. 3 A：In STM sites, for upstream sgRNA1, cutting feelings of the Cas9 and Cas9 mutant to target DNA fragment Condition.
Fig. 3 B：In STM sites, for downstream sgRNA2, cutting feelings of the Cas9 and Cas9 mutant to target DNA fragment Condition.
Embodiment
First, Cas9 nucleases
The Cas9 nucleases (Cas9-F916P) of the present invention, with Cas9 nucleases, it is adaptable to CRISPR/Cas9 systems System, the Cas9 nucleases are that the 916th phenylalanine of wild type Cas9 nucleases is mutated into proline to obtain.The Cas9 Nuclease (Cas9-F916P) is produced compared with wild type Cas9 nucleases when being cut to target gene group DNA fragmentation The ratio of prominent broken ends and blunt broken ends is different.Further, the wild type Cas9 nucleases are SpCas9.Enter one Step ground, the amino acid sequence of the wild type Cas9 nucleases is as shown in SEQ ID NO.7.
Further, the Cas9 nucleases (Cas9-F916P) contain the amino acid sequence as shown in SEQ ID NO.9, In some embodiments of the present invention, the amino acid sequence of the Cas9-F916P is illustrated as shown in SEQ ID NO.9.
2nd, the polynucleotides of Cas9 nucleases are encoded
The polynucleotides of the Cas9 nucleases (Cas9-F916P) are encoded, can be DNA form or rna form.DNA shapes Formula includes cDNA, genomic DNA or artificial synthesized DNA.DNA can be single-stranded or double-strand.
The polynucleotides of the Cas9 nucleases (Cas9-F916P) are encoded, can be by well known to those skilled in the art It is prepared by any appropriate technology.The technology sees the general description of this area, such as《Molecular Cloning:A Laboratory guide》(J. Pehanorm cloth Luke etc., Science Press, 1995).The including but not limited to method such as recombinant DNA technology, chemical synthesis.
In some embodiments of the present invention, the polynucleotides for encoding the Cas9 nucleases (Cas9-F916P) are illustrated As shown in SEQ ID NO.10.
3rd, expression vector
The expression vector contains the polynucleotides for encoding the Cas9 nucleases (Cas9-F916P).The technology of this area Method known to personnel can be used to build the expression vector.These methods include recombinant DNA technology, DNA synthetic technologys etc..Can The DNA of the Cas9 nucleases (Cas9-F916P) is effectively connected in the multiple cloning sites in carrier, to instruct mRNA to close Into so expressing protein.
4th, host cell
The expression vector that the host cell is expressed the Cas9 nucleases (Cas9-F916P) is converted.Host cell Can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast cells；Or higher eucaryotic cells, such as lactation Zooblast.Representative example has:Escherichia coli, streptomyces；Salmonella typhimurtum, Liszt bacterium；Fungal cell's such as ferment It is female；Plant cell；Drosophila S2 or Sf9 insect cell；The zooblast of CHO, COS.293 cell or Bowes melanoma cells Deng.
5th, the method for preparing Cas9 nucleases (Cas9-F916P)
The method for preparing foregoing Cas9 nucleases (Cas9-F916P), including step：Build and contain Cas9 nucleases (Cas9-F916P) expression vector of encoding polynucleotide sequence, then converts into host cell induction by the expression vector Expression, separation obtains described Cas9 nucleases (Cas9-F916P) from expression product.
Those skilled in the art can select suitable expression vector according to the property of Cas9 nucleases (Cas9-F916P) And host cell.
6th, Cas9 nucleases (Cas9-F916P) or its coded polynucleotide or the expression containing the coded polynucleotide The purposes of carrier
The Cas9 nucleases (Cas9-F916P) or its coded polynucleotide of the present invention contain the coded polynucleotide Expression vector can be used for genomic DNA fragment editor or for preparing genomic DNA fragment edit tool.
Further, the editor includes unit point editor and many sites editor.The editing sites of many site editors Number for two and more than.The mode of the editor includes mutation, deletes, inverts or inversion, repetition, transposition or insertion.
7th, genomic DNA fragment edit tool
The genomic DNA fragment edit tool of the present invention can be CRISPR/Cas9 systems, the CRISPR/Cas9 systems System includes foregoing Cas9 nucleases (Cas9-F916P) or its coded polynucleotide or the expression containing the coded polynucleotide Carrier.Further, the CRISPR/Cas9 systems also include one or more sgRNA for target DNA fragment.It is described SgRNA is for designed by target DNA fragment, under sgRNA (Single-guide RNA) mediation, Cas9-F916P can DNA double chain is cut in PAM (Protospacer adjacent motif) site upstream, DNA double chain fracture is formed, leads to Cell itself repair system is crossed, the accurate editor of DNA fragmentation is completed.SgRNA for target gene can be one or two And more than.When sgRNA is one, it is possible to achieve to the unit point editor of target DNA fragment, when sgRNA be two and When the above, it is possible to achieve many sites of target DNA fragment are edited.
8th, genomic DNA fragment edit methods
The genomic DNA fragment edit methods of the present invention, using foregoing Cas9 nucleases (Cas9-F916P) and therewith The one or more sgRNA coordinated, edlin is entered using CRISPR/Cas9 systems to genomic DNA fragment to be edited.It is described Editor includes unit point editor and many sites editor.The edit bit points of many sites editor for two and more than.Work as sgRNA , can be with when sgRNA is two and the above when being one, it is possible to achieve to the unit point editor of target DNA fragment Realize and many sites of target DNA fragment are edited.Further, foregoing Cas9 nucleases (Cas9-F916P) can be encoded multinuclear The expression vector of thuja acid and matched one or more sgRNA are together transferred in cell, to genomic DNA to be edited Fragment enters edlin.
9th, genomic DNA fragment unit point edit methods
Using CRISPR/Cas9 systems, DNA double chain is cut using the Cas9 nucleases (Cas9-F916P) of the present invention The prominent broken ends of generation are cut, the base complementary with prominent broken ends is added in the way of filling-in is connected, be can be achieved to gene The unit point editor of group DNA fragmentation.Base when the genomic DNA fragment unit point edit methods can change unit point editor The feature of mutation.
The filling-in connection refers to：The prominent broken ends can first pass through base pair complementarity and add and prominent end Complementary base filling-in is reconnection after blunt ends.
As enumerated in some embodiments of the present invention, Cas9 nucleases F916P is under sgRNA1 mediation, to base When being cut because of a group DNA fragmentation (β-globin RE2 sites), produced protrusion broken ends U4, in cell repair system In the presence of, the prominent broken ends U4 can first pass through base pair complementarity and add the bases G benefit complementary with prominent end C Put down to be connected with jointing again after blunt ends.
Cas9 nucleases F916P is carried out under sgRNA2 mediation to genomic DNA fragment (β-globin RE2 sites) During cutting, produced protrusion broken ends D4, in the presence of cell repair system, the prominent broken ends D4 can first lead to Base pair complementarity is crossed to add the base T filling-in with prominent end A complementations to be connected with jointing again after blunt ends.
Explanation：
In the present invention, Cas9 can be used as the abbreviation of Cas9 nucleases, be looked like identical with Cas9 nucleases.In this hair In bright, alternatively used between Cas9-F916P, F916P, F916P mutant, the meaning is entitled F916P Cas9 nucleic acid Enzyme.
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention.The test method of unreceipted actual conditions in the following example, Generally according to normal condition, or according to the condition proposed by each manufacturer.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range Any one numerical value can select between point and two end points.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except the specific method used in embodiment, equipment, Outside material, according to those skilled in the art to the grasp of prior art and the record of the present invention, it can also use and this Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.
Unless otherwise indicated, disclosed in this invention experimental method, detection method, preparation method using this technology lead Domain conventional molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987 and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The connection of the researching DNA part edit joint of embodiment 1 finds Cas9 cutting new mechanisms
For HS51 sites, the sgRNAs plasmids for HS51 sites are built：
(1) primer is bought
Bought from Shanghai Sani bio tech ltd and target having for sequence for the sgRNAs of HS51 sites sum respectively 5 ' suspended ends " ACCG " and " AAAC " can be with forward and reverse deoxy-oligonucleotides of complementary pairing；
Sequence is targetted for the sgRNAs in above-mentioned HS51 sites：
HS51RE1sgRNA1：GCCACACATCCAAGGCTGAC(SEQ ID NO.1)
HS51RE1sgRNA2：GAGATTTGGGGCGTCAGGAAG(SEQ ID NO.2)
(2) double-stranded DNA with suspended end of complementary pairing is obtained
1) ddH is used2Deoxy-oligonucleotide is dissolved to 100 μM by O, and is diluted to 20 μM；
2) positive and negative deoxy-oligonucleotide is added into following reaction system：
Reaction condition：Then 95 DEG C of water-baths, 5min opens water-bath lid temperature and is down to 60 DEG C or so, close the lid cold But to room temperature.
(3) digestion pGL3-U6-sgRNA-PGK-Puro vector
1) BsaI digestion with restriction enzyme vector plasmids are used, reaction system is as follows：
Reaction condition：37 DEG C, 1.5 hours；
2) glue reclaim purified dnase section section, illustrates purifying according to glue reclaim kit (Axygen).
(4) carrier after connection digestion and the double-stranded DNA with suspended end
Linked system is as follows：
Reaction condition：Room temperature reaction 1.5 hours；
(5) connection product is converted
Connection product is converted with Stbl3 competence, in the antibiotic of benzyl containing ammonia (Amp, 100mg/L) LB plate incubated overnights, 37℃。
(6) picking monoclonal is sequenced
1) from picking single bacterium colony on ammonia benzyl antibiotic LB flat boards, LB (Amp, 100mg/L) Liquid Culture is stayed overnight；
2) plasmid extraction, illustrates extraction according to the small kit (Axygen) of taking out of plasmid；
3) plasmid after extracting serves extra large Sani bio tech ltd sequencing.
(7) successfully plasmid is sequenced to take out in carrying out
1) successful plasmid is sequenced to be converted again with Stbl3 competence, in the LB flat board cultures containing Amp (100mg/L) Night；
2) morning picking single bacterium colony is cultivated 8 hours in 2ml LB (Amp, 100mg/L) fluid nutrient medium, is then transferred to Overnight incubation in 200ml LB (Amp, 100mg/L) fluid nutrient medium；
3) bacterium is collected, illustrates to extract plasmid according to kit (Qiagen) is taken out in plasmid.
2. it is prepared by humanization Cas9 plasmids
1) humanization Cas9 plasmids are built middle laboratory from Peking University's seat and obtained；
2) converted again with Stbl3 competence, in LB flat boards (Amp, 100mg/L) overnight incubation；
3) morning picking single bacterium colony is cultivated 8 hours in 2ml LB (Amp, 100mg/L) fluid nutrient medium, is then transferred to Overnight incubation in 200ml LB (Amp, 100mg/L) fluid nutrient medium, taken out in plasmid.
3. carry out cell transfecting with Lipofectamine 2000
1) HEK293T cell culture, at 37 DEG C, contains 5%CO in blake bottle2Cultivated in cell culture incubator, treat that it is grown To blake bottle 80~90%.
2) cell grown (is added into 10% hyclone, without blue or green chain in 12 orifice plates with the complete nonreactive culture mediums of DMEM Mycin is dual anti-) carry out bed board, incubated overnight.
3) when the cell length in 12 orifice plates is to 80~90%, by the humanization Cas9 plasmids (800ng) and pin that prepare Cell transfecting is carried out by Lipofectamine 2000 to the sgRNAs plasmids (each 600ng) in HS51 sites, each sample is each Two repetitions.
4) transfect after two days, collect cell, with genome extracts kit (Genomic DNA Purification kit, Promega) extract genome.
4. prepare high-throughput sequencing library
It is expected to design primer at deletion, reversion and the accurate connection site upstream about 30bp for repeating joint in DNA fragmentation, Primer 5 ' is then held to the sequence measuring joints for adding the Illumina with barcode, anti-sense primer can be designed away from splicing Site some position and plus Illumina sequence measuring joints, enter performing PCR amplification, then using Roche PCR purification kits (Product No.:11732676001) purified, DNA product is dissolved in 10mM Tris-HCL buffer (PH=8.5), Storehouse is formed after mixed in equal amounts, high-flux sequence is carried out.
High flux primer：
Hiseq-hHs51-aF:
ATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGCAAGGAGATCCGTGTCGTC (SEQ ID NO.3)
Hiseq-hs51-aRa：
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGGATGTTGTGGAAGGCG AGCAG(SEQ ID NO.4)
Hiseq-hs51-bFa:
CAAGCAGAAGACGGCATACGAGATGGACGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTTTACATGACAG CTTCCGGTAG(SEQ ID NO.5)
Hiseq-hHs51-bR:
CAAGCAGAAGACGGCATACGAGATTTGACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTTTTGGCTAACA ACATAGTGCTTC(SEQ ID NO.6)。
5. high-flux sequence data processing
After the completion of high-flux sequence, the sequencing result of sample is separated from library by barcode using Linux programs Come, be stored in respective file, then carry out BWA-MEM comparisons, the sequence after comparison passes through Varscan2 programs (V2.3.9) insertion and deletion mutation of analysis DNA fragmentation, Varscan2 program parameters are as follows：
The present invention has found that Cas9 cuts new mechanism by the end connection of researching DNA part edit.
As shown in Figure 1A, combined using the sgRNA of two sgRNAs formation and Cas9 nucleases enter to genomic DNA fragment During edlin, Cas9 nucleases carry out cutting to genomic DNA double-strand under two sgRNAs mediations and produce four broken ends (DSB), these broken ends (DSB) produced in the presence of cell repair system (for example, MRN/CtIP) DNA fragmentation delete, The DNA fragmentation editor such as reversion and repetition.
As shown in Figure 1B, for genomic DNA fragment HS51RE1 (HS51 sites), we use sgRNA1 and sgRNA2 The sgRNA combinations of formation and Cas9 nucleases enter edlin to it.Then, we detect DNA fragmentation delete, reversion and again It is multiple, the deletion of recycling high throughput sequencing technologies detection DNA fragmentation, reversion and the situation for repeating jointing, except with being expected phase Outside, DNA fragmentation deletes jointing, reversion downstream connection joint and repeats to connect for the accurate connection (Joined precisely) of symbol Connect joint and all there is a certain proportion of base addition (Insertion).
As shown in Figure 1 C, detect that DNA fragmentation deletes the situation of jointing using high throughput sequencing technologies, be consistent with expection Accurate connection (Joined precisely) ratio account for 79.23%, delete the addition that joint also has " G " base (Insertion, compared with expected accurate connection), its ratio accounts for 11.13%.
Compared with expected accurate connection, thus it is speculated that DNA fragmentation deletes " G " base added at jointing and is derived from mould The bases of version DNA (HS51RE1, HS51 site) PAM upstreams 3bp nearby (specially at the 4bp of PAM upstreams).Thus it is speculated that When the Cas9 nucleases pair DNA complementary with sgRNA is cut, cut at the 3bp of PAM upstreams；And Cas9 nucleic acid When the DNA of enzyme pair and sgRNA incomplementarities is cut, cut at 4bp that can be farther at the 3bp of PAM upstreams.According to DNA fragmentation deletes the addition that there is " G " base at jointing (compared with expected accurate connection), thus it is speculated that Cas9 nucleases exist When being cut under sgRNA2 mediations to genomic DNA fragment, there are blunt ends cutting and protruding terminus cutting, and then produce different Broken ends.When Cas9 nucleases have carried out blunt ends cutting under sgRNA2 mediations to genomic DNA fragment, that is, The DNA of the Cas9 nucleases pair DNA complementary with sgRNA and incomplementarity when cutting is carried out at the 3bp of PAM upstreams Cutting, generates blunt broken ends " E3 ".Blunt broken ends " E3 " produce DNA fragmentation deletion in the presence of cell repair system When, DNA fragmentation will not be caused to delete the addition of " G " base at jointing, but produce accurate connection in line (Joined precisely).Cut when Cas9 nucleases have carried out protruding terminus under sgRNA2 mediations to genomic DNA fragment When cutting, that is, Cas9 nucleases pair and sgRNA complementary DNA when cutting are cut at the 3bp of PAM upstreams, And when pair being cut with the DNAs of sgRNA incomplementarities cut at the 4bp of PAM upstreams, so that it is prominent disconnected to generate 5 ' Split end " E4 ".5 ' prominent broken ends " E4 " can cause when producing DNA fragmentation deletion in the presence of cell repair system DNA fragmentation deletes the addition of " G " base at jointing.
It is therefore believed that：Under the cutting of Cas9 nucleases, in the broken ends of generation, blunt broken ends E3 ratio Ratio=79.23% of example=expected accurate connection (Joined precisely) being consistent.Prominent broken ends E4 ratio Additional proportion=11.13% of=" G " bases.
But, it is observed that except accurate connection (Joined precisely) in line and DNA fragmentation Delete at jointing beyond this two major classes situation of the addition of presence " G " base, the random base of an also class deletes (Small deletion).It is considered that it is each broken ends (blunt broken ends E3 that this kind of random base, which deletes (Small deletion), With prominent broken ends E4) randomly generated in the presence of cell repair system, each broken ends are produced with impartial probability Raw base deletes (Small deletion), and base of each broken ends produced by the presence of cell repair system is deleted The quantity of (Small deletion) is directly proportional to the quantity of each broken ends.
The presence of phenomenon is deleted based on randomized bases, it is believed that, the actual measurement ratio of each broken ends obtained by sequencing There is gap, it is necessary to be modified reduction in example, i.e., with its actual proportions on the basis of the actual measurement ratio sum of various broken ends, The ratio of each broken ends is calculated, in this, as the accounting of the broken ends.I.e. to each produced by the cutting of Cas9 nucleases The ratio of broken ends is standardized calculating, and blunt broken ends E3 ratio is 87.7%【Computational methods are：79.23% ÷ (79.23%+11.13%)】.Prominent broken ends E4 ratio is 12.3%【Computational methods are：11.13% ÷ (79.23%+ 11.13%)】.That is, in the cutting mode that Cas9 nucleases are carried out under sgRNA2 mediation to genomic DNA fragment, blunt end The ratio of end cutting is 87.7%, and the ratio of protruding terminus cutting is 12.3%.
As shown in figure iD, detect that DNA fragmentation repeats the situation of jointing using high throughput sequencing technologies, be consistent with expection Accurate connection (Joined precisely) ratio account at 8.96%, jointing exist " T " base addition The ratio of (Insertion, compared with expected accurate connection) accounts for 82.92%.
Compared with expected accurate connection, thus it is speculated that " T " base that DNA fragmentation repeats to add at jointing is derived from mould The bases of PAM upstreams 3bp nearby (specially at the 4bp of PAM upstreams) on version DNA (HS51RE1, HS51 site).Thus it is speculated that When the Cas9 nucleases pair DNA complementary with sgRNA is cut, cut at the 3bp of PAM upstreams；And Cas9 nucleic acid When the DNA of enzyme pair and sgRNA incomplementarities is cut, cut at 4bp that can be farther at the 3bp of PAM upstreams.According to DNA fragmentation repeats to detect the presence of the addition of " T " base at jointing (compared with expected accurate connection), thus it is speculated that Cas9 cores When sour enzyme is cut under sgRNA1 mediations to genomic DNA fragment, there are blunt ends cutting and protruding terminus cutting, and then produce Raw different broken ends.When Cas9 nucleases have carried out blunt ends cutting under sgRNA1 mediations to genomic DNA fragment, It is exactly that Cas9 nucleases pair are at the 3bp of PAM upstreams when being cut with the DNA of sgRNA complementations and the DNA of incomplementarity Cut, generate blunt broken ends " C3 ".Blunt broken ends " C3 " produce DNA fragmentation in the presence of cell repair system When repeating, DNA fragmentation will not be caused to repeat the addition of " T " base at jointing, but produce and in line precisely connect Connect (Joined precisely).When Cas9 nucleases have carried out protruding terminus under sgRNA1 mediations to genomic DNA fragment During cutting, that is, Cas9 nucleases pair and sgRNA complementary DNA when cutting are cut at the 3bp of PAM upstreams When cutting, and pair being cut with the DNAs of sgRNA incomplementarities cut at the 4bp of PAM upstreams, dashed forward so as to generate 5 ' Go out broken ends " C4 ".5 ' prominent broken ends " C4 " can lead when producing DNA fragmentation repetition in the presence of cell repair system DNA fragmentation is caused to repeat the addition of " T " base at jointing.
It is therefore believed that：Under the cutting of Cas9 nucleases, in the broken ends of generation, blunt broken ends C3 ratio Ratio=8.96% of example=expected accurate connection (Joined precisely) being consistent.Prominent broken ends C4 ratio= Additional proportion=82.92% of " T " base.
But, it is observed that except accurate connection (Joined precisely) in line and DNA fragmentation Repeatedly at jointing beyond this two major classes situation of the addition of presence " T " base, the random base of an also class deletes (Small deletion).It is considered that it is each broken ends (blunt broken ends C3 that this kind of random base, which deletes (Small deletion), With prominent broken ends C4) randomly generated in the presence of cell repair system, each broken ends are produced with impartial probability Raw base deletes (Small deletion), and base of each broken ends produced by the presence of cell repair system is deleted The quantity of (Small deletion) is directly proportional to the quantity of each broken ends.
The presence of phenomenon is deleted based on randomized bases, it is believed that, the actual measurement ratio of each broken ends obtained by sequencing There is gap, it is necessary to be modified reduction in example, i.e., with its actual proportions on the basis of the actual measurement ratio sum of various broken ends, The ratio of each broken ends is calculated, in this, as the accounting of the broken ends.I.e. to each produced by the cutting of Cas9 nucleases The ratio of broken ends is standardized calculating, and blunt broken ends C3 ratio is 9.75%【Computational methods are：8.96% ÷ (8.96%+82.92%)】.Prominent broken ends C4 ratio is 90.25%【Computational methods are：82.92% ÷ (8.96%+ 82.92%)】.That is, in the cutting mode that Cas9 nucleases are carried out under sgRNA1 mediation to genomic DNA fragment, blunt end The ratio of end cutting is 9.75%, and the ratio of protruding terminus cutting is 90.25%.
As referring to figure 1E, according to Cas9 nucleases under sgRNA1 and sgRNA2 mediation respectively to genomic DNA fragment The mode ratio cut, predicts the sequence of the broken ends produced, and then extrapolates DNA fragmentation reversion downstream connection joint The base at place adds situation and ratio.
When Cas9 nucleases carry out protruding terminus cutting under sgRNA1 mediation to genomic DNA fragment, protrusion is produced Broken ends " C4 ", Cas9 nucleases carry out blunt ends cutting under sgRNA2 mediation to genomic DNA fragment, produce blunt disconnected End " E3 " is split, then in the presence of cell repair system, the addition of " A " base occurs at DNA fragmentation reversion downstream tap, And the ratio occurred is 79.14%【Computational methods are：" C4 " protrudes broken ends accounting (90.25%) x " E3 " blunt broken ends Accounting (87.7%)=79.14%】, with testing " A " base additional proportion at the DNA fragmentation detected reversion downstream tap 71.94% is close.
When Cas9 nucleases carry out blunt ends cutting under sgRNA1 mediation to genomic DNA fragment, blunt fracture is produced End " C3 ", Cas9 nucleases carry out protruding terminus cutting under sgRNA2 mediation to genomic DNA fragment, produce prominent disconnected End " E4 " is split, then in the presence of cell repair system, the addition of " G " base occurs at DNA fragmentation reversion downstream tap, And the ratio occurred is 1.19%【Computational methods are：The prominent broken ends of " C3 " blunt broken ends accounting (9.75%) x " E4 " are accounted for Than (12.3%)=1.19%】, with testing " G " base additional proportion 8.54% at the DNA fragmentation detected reversion downstream tap It is close.
When Cas9 nucleases carry out protruding terminus cutting under sgRNA1 mediation to genomic DNA fragment, protrusion is produced Broken ends " C4 ", Cas9 nucleases carry out protruding terminus cutting under sgRNA2 mediation to genomic DNA fragment, produce prominent Go out broken ends " E4 ", then in the presence of cell repair system, " AG " base occurs at DNA fragmentation reversion downstream tap Add, and the ratio occurred is 11%【Computational methods are：The prominent fracture of " C4 " prominent broken ends accounting (90.25%) x " E4 " End accounting (12.3%)=11%】, with testing " AG " base additional proportion at the DNA fragmentation detected reversion downstream tap 3.66% is close.
When Cas9 nucleases carry out blunt ends cutting under sgRNA1 mediation to genomic DNA fragment, blunt fracture is produced End " C3 ", Cas9 nucleases carry out blunt ends cutting under sgRNA2 mediation to genomic DNA fragment, produce blunt fracture end Hold " E3 ", then in the presence of cell repair system, DNA fragmentation reversion downstream tap is precisely connected, and the ratio occurred is 8.55%【Computational methods are：The blunt broken ends accounting (87.7%) of " C3 " blunt broken ends accounting (9.75%) x " E3 "= 8.55%】, precisely it is connected ratio 6.67% with the DNA fragmentation reversion downstream tap that experiment is detected close.
In summary, Fig. 1 E experimental result further demonstrate：The DNA of Cas9 nucleases pair and sgRNA incomplementarities , can be at the 3bp of PAM upstreams to being cut at farther base when being cut.Cas9 nucleases are under sgRNA mediations to gene When group DNA fragmentation is cut, there are blunt ends cutting and protruding terminus cutting, and then produce different broken ends.These fractures End produces accurate DNA fragmentation editor (the accurate volume of particular bases in line in the presence of cell repair system Volume) or with the expected gene editing not being inconsistent (random base is deleted).
As shown in fig. 1F, in sgRNA combinations, sgRNA design is different (target sequence is different), and Cas9 nucleases are in sgRNA Mediation under carry out that cutting mode ratio is different to genomic DNA fragment, the broken ends ratio of generation is different.Specifically, When Cas9 nucleases are cut under sgRNA1 mediation to genomic DNA fragment, the accounting of blunt ends cutting mode is higher than Protruding terminus cutting mode accounting, the blunt broken ends accounting of generation is higher than 5 ' prominent broken ends accountings.But Cas9 nucleic acid When enzyme is cut under sgRNA2 mediation to genomic DNA fragment, the accounting of protruding terminus cutting mode is higher than blunt ends Cutting mode accounting, 5 ' prominent broken ends accountings of generation are also above blunt broken ends accounting.Because it is found that Cas9 nucleases The mode cut under sgRNA mediations to genomic DNA fragment has blunt ends to cut and protruding terminus cutting, when Cas9 cores Sour enzyme carries out protruding terminus cutting under sgRNA mediations to genomic DNA fragment, when producing prominent broken ends, according to filling-in The mode of connection can add the base complementary with prominent broken ends, so as to realize the alkali to genomic DNA fragment ad-hoc location Base is added.
The specific Cas9 that the mutation of embodiment 2 SpCas9 obtains cutting mode change realizes accurately DNA fragmentation editor
1. build Cas9 mutant
1) Cas9 is built using NEB mutagenesis kits (Q5Site-Directed Mutagenesis Kit, #E0554S) Mutant, enters performing PCR amplification, reacts as follows first：
2) KLD (Kinase, Ligase＆DpnI) is handled, and is reacted as follows：
Reaction condition：Room temperature 10 minutes
3) reaction product in 2) is completely used for competence bacterium Stbl3 (50 μ l) conversion, in the antibiotic of benzyl containing ammonia (Amp, 100mg/L) LB plate incubated overnights, 37 DEG C.Sequencing is sent after picking monoclonal, plasmid extraction.SpCas9's (Cas9WT) Amino acid sequence is as shown in SEQ ID NO.7, specially：
SpCas9 (Cas9WT) coding nucleotide sequence is as shown in SEQ ID NO.8, specially：
As shown in Figure 2 A, Cas9 nucleases, containing RuvC and HNH functional domains, it is non-with sgRNA that RuvC functional domains are responsible for cutting Complementary DNA, HNH functional domains are responsible for the cutting DNA complementary with sgRNA.
Claimed Cas9 nucleic acid enzyme mutants are named as Cas9-F916P (by SpCas9 nucleases the 916th Position phenylalanine is mutated into proline acquisition),
Shown in Cas9-F916P amino acid sequence SEQ ID NO.9, it is specially：
Cas9-F916P coding nucleotide sequence is as shown in SEQ ID NO.10, specially：
In addition, to carry out mutant K775A, R778A, E779A, K918P of random mutation acquisition to SpCas9 as right According to these control mutant are different from the Cas9-F916P of present invention sequence.
2.Cas9 nucleic acid enzyme mutant carries out DNA fragmentation editor
(1) β-globin RE2 (RRM21 sites) are directed to, the sgRNAs of RRM21 sites (β-globin RE2) is built.
The sgRNAs targets sequence：
β-globin RE2sgRNA1：ACCCAATGACCTCAGGCTGT(SEQ ID NO.11)；
β-globin RE2sgRNA2：TCACTTGTTAGCGGCATCTG(SEQ ID NO.12)；
The sgRNAs targetings for β-globin RE2 (RRM21 sites) are bought from Shanghai Sani bio tech ltd Sequence has 5 ' suspended ends " ACCG " and " AAAC " can be with forward and reverse deoxy-oligonucleotide of complementary pairing.
(2) double-stranded DNA with suspended end of complementary pairing is obtained
1) ddH is used2Deoxy-oligonucleotide is dissolved to 100 μM by O, and is diluted to 20 μM；
2) positive and negative deoxy-oligonucleotide is added into following reaction system：
Reaction condition：Then 95 DEG C of water-baths, 5min opens water-bath lid temperature and is down to 60 DEG C or so, close the lid cold But to room temperature.
(3) digestion pGL3-U6-sgRNA-PGK-Puro vector
1) BsaI digestion with restriction enzyme vector plasmids are used, reaction system is as follows：
Reaction condition：37 DEG C, 1.5 hours；
2) glue reclaim purified dnase section section, illustrates purifying according to glue reclaim kit (Axygen).
(4) carrier after connection digestion and the double-stranded DNA with suspended end
Linked system is as follows：
Reaction condition：Room temperature reaction 1.5 hours；
(5) connection product is converted
Connection product is converted with Stbl3 competence, in the antibiotic of benzyl containing ammonia (Amp, 100mg/L) LB plate incubated overnights, 37℃。
(6) picking monoclonal is sequenced
1) from picking single bacterium colony on ammonia benzyl antibiotic LB flat boards, LB (Amp, 100mg/L) Liquid Culture is stayed overnight；
2) plasmid extraction, illustrates extraction according to the small kit (Axygen) of taking out of plasmid；
3) plasmid after extracting serves extra large Sani bio tech ltd sequencing.
(7) successfully plasmid is sequenced to take out in carrying out
1) successful plasmid is sequenced to be converted again with Stbl3 competence, in the LB flat board cultures containing Amp (100mg/L) Night；
2) morning picking single bacterium colony is cultivated 8 hours in 2ml LB (Amp, 100mg/L) fluid nutrient medium, is then transferred to Overnight incubation in 200ml LB (Amp, 100mg/L) fluid nutrient medium；
3) bacterium is collected, illustrates to extract plasmid according to kit (Qiagen) is taken out in plasmid.
(8) cell transfecting is carried out with Lipofectamine 2000
1) HEK293T cell culture, at 37 DEG C, contains 5%CO in blake bottle2Cultivated in cell culture incubator, treat that it is grown To blake bottle 80~90%, the cell grown is subjected to bed board, incubated overnight in 12 orifice plates with the complete nonreactive culture mediums of DMEM；
2) when the cell length in 12 orifice plates is to 80~90%, by Cas9 the and Cas9 mutant plasmids prepared (800ng) carries out cell by Lipofectamine 2000 with the sgRNAs plasmids (each 600ng) for RRM21 sites and turned Dye, each each two repetitions of sample.
3) transfect after two days, collect cell, with genome extracts kit (Genomic DNA Purification kit, Promega) extract genome.
(9) high-throughput sequencing library is prepared
It is expected to design primer at deletion, reversion and the accurate connection site upstream about 30bp for repeating joint in DNA fragmentation, Primer 5 ' is then held to the sequence measuring joints for adding the Illumina with barcode, anti-sense primer can be designed away from splicing Site some position and plus Illumina sequence measuring joints, enter performing PCR amplification, then using Roche PCR purification kits (Product No.:11732676001) purified, DNA product is dissolved in 10mM Tris-HCL buffer (PH=8.5), Storehouse is formed after mixed in equal amounts, high-flux sequence is carried out.
Cas9 mutant primers：
Cas9-F916P-F：TAAAGCAGGCcccATCAAAAGGCAG(SEQ ID NO.13)；
Cas9-F916P-R：TCCAACTCAGACAGGCCA(SEQ ID NO.14)；
(10) high-flux sequence data processing
After the completion of high-flux sequence, the sequencing result of sample is separated from library by barcode using Linux programs Come, be stored in respective file, then carry out BWA-MEM comparisons, the sequence after comparison passes through Varscan2 programs (V2.3.9) insertion and deletion mutation of analysis DNA fragmentation, Varscan2 program parameters are as follows：
For β-globin RE2 sites, using high-flux sequence primer carry out the deletion of pcr amplified DNA fragment, reversion and Repeat, build storehouse and carry out high-flux sequence.
High flux primer：
Hiseq-RRM-1F3：
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTATATGGCATCCTAGCCTTA AGAAACTAG(SEQ ID NO.15)
Hiseq-RRM-1R2：
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTACGACGCAGGAGCCGTAT CATG(SEQ ID NO.16)
Hiseq-RRM-3F2：
CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATAGCAATGAAAT CTTGAAGGAGTG(SEQ ID NO.17)
Hiseq-RRM-3R2：
CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCACAGCCCTGCT CTATTACG(SEQ ID NO.18)。
With reference to the method for above-described embodiment 1, combined using the sgRNA of two sgRNAs formation and Cas9 nucleases are to gene Group DNA fragmentation enters after edlin, detects that the jointing base that DNA fragmentation is deleted and repeated adds using high throughput sequencing technologies Enter situation, and then calculate Cas9 nucleases when being cut under each sgRNA is mediated to genomic DNA fragment, blunt ends are cut Cut the accounting of mode and protruding terminus cutting mode.
Specifically, wild type SpCas9 nucleases (abbreviation Cas9WT, WT) (Fig. 2A) and F916P are each in sgRNA combinations Enter two sgRNAs of edlin schematic diagram such as Fig. 2 B under sgRNA mediations to genomic DNA fragment β-globin RE2 sites.
As shown in Figure 2 C, using high throughput sequencing technologies detect DNA fragmentation repeat jointing situation, except with expection Beyond the accurate connection (Joined precisely) being consistent, also exist compared with expected accurate connection, add at jointing The situation of " C " base and " GC " base is entered.During from different Cas9 nucleases, what is detected in line precisely connects Connect (Joined precisely), "+C " base, the accounting of "+GC " base difference.Using from this Cas9 nuclease of F916P as Example, the accounting for detecting accurate connection (Joined precisely) in line is 38.04%, the accounting of "+C " base For 22.70%, the accounting of "+GC " base is 11.99%.
In view of DNA fragmentation repeats to detect the presence of the addition of " C " base (with expected accurate connection phase at jointing Than), it is presumed that " C " base that DNA fragmentation repeats to add at jointing is derived from masterplate DNA (β-globin RE2 Point) on PAM (AGG) upstream 4bp at base.Also, further speculate Jie of this Cas9 nuclease of F916P in sgRNA1 Lead lower when being cut to genomic DNA fragment (β-globin RE2 sites), a pair DNA complementary with sgRNA is cut When, be to be cut at the 3bp of PAM upstreams, and pair with sgRNA incomplementarity DNAs cut when, then be on PAM (AGG) Swim and protruding terminus cutting is carried out at 4bp, so as to generate prominent broken ends U4.Prominent broken ends U4 is in cell repair system In the presence of produce DNA fragmentation repeat when, result in DNA fragmentation repeat jointing at " C " base addition.
Similarly, in view of the addition that DNA fragmentation repeats to detect the presence of " GC " base at jointing (precisely connects with expected Connect and compare), it is presumed that " GC " base that DNA fragmentation repeats to add at jointing is derived from masterplate DNA (β-globin RE2 sites) on PAM (AGG) upstream 4bp at and 5bp base.Further speculate that this Cas9 nuclease of F916P exists When being cut under sgRNA1 mediation to genomic DNA fragment (β-globin RE2 sites), couple DNA complementary with sgRNA When chain is cut, cut at the 3bp of PAM upstreams, and pair with sgRNA incomplementarity DNAs cut when, be Protruding terminus cutting is carried out at PAM (AGG) upstream 5bp, so as to generate prominent broken ends U5.Prominent broken ends U5 is thin In the presence of born of the same parents' repair system produce DNA fragmentation repeat when, result in DNA fragmentation repeat jointing at " GC " base plus Enter.
And when F916P this Cas9 nuclease under sgRNA1 mediation to genomic DNA fragment (β-globin RE2 Point) when a pair DNA complementary with sgRNA is cut, cut at the 3bp of PAM upstreams when being cut, pair and It is the progress blunt ends cutting at PAM (AGG) upstream 3bp when sgRNA incomplementarity DNAs are cut, so as to generate blunt disconnected Split end U3.Blunt broken ends U3 will not cause DNA fragmentation when producing DNA fragmentation repetition in the presence of cell repair system The addition of base at jointing is repeated, but produces accurate connection (Joined precisely) in line.
It is therefore believed that：Under Cas9 nucleases F916P cutting, in the broken ends of generation, blunt broken ends U3 Ratio=38.04% of accurate connection (Joined precisely) that is consistent of accounting=expection.Prominent broken ends U4's Additional proportion=22.70% of ratio=" C " base.The additional proportion of prominent broken ends U5 ratio=" GC " base= 11.99%.
But, it is observed that except accurate connection (Joined precisely) in line, " C " base Add and the addition of " GC " base this three major types situation beyond, the random base of an also class deletes (Small deletion).It is considered that it is each broken ends (blunt broken ends that this kind of random base, which deletes (Small deletion), U3/ protrudes broken ends U4/ and protrudes broken ends U5) randomly generated in the presence of cell repair system, each broken ends Base is produced with impartial probability and deletes (Small deletion), each broken ends institute in the presence of cell repair system The quantity that the base of generation deletes (Small deletion) is directly proportional to the quantity of each broken ends.
The presence of phenomenon is deleted based on randomized bases, it is believed that, the actual measurement ratio of each broken ends obtained by sequencing There is gap, it is necessary to be modified reduction in example, i.e., with its actual proportions on the basis of the actual measurement ratio sum of various broken ends, The ratio of each broken ends is calculated, in this, as the accounting of the broken ends.I.e. to produced by Cas9 nucleases F916P cutting The accountings of each broken ends be standardized calculating, blunt broken ends U3 accounting is 52.30%【Computational methods are： 38.04% ÷ (38.04%+22.70%+11.99%)】.Prominent broken ends U4 ratio is 31.21%【Computational methods are： 22.70% ÷ (38.04%+22.70%+11.99%)】.Prominent broken ends U5 ratio is 16.49%【Computational methods are： 11.99% ÷ (38.04%+22.70%+11.99%)】.
That is, in the cutting mode that Cas9 nucleases F916P is carried out under sgRNA1 mediation to genomic DNA fragment, The ratio of U3 blunt ends cutting is that the ratio of 52.30%, U4 protruding terminuses cutting is the ratio of 31.21%, U5 protruding terminuses cutting Example is 16.49%.
With reference to the above method, then to calculate wild type Cas9 nucleases (abbreviation Cas9WT, WT) right under sgRNA1 mediation In the cutting mode that genomic DNA fragment is carried out, the cutting of accounting X1, U4 protruding terminus X2, U5 of the cutting of U3 blunt ends protrude end Hold the accounting X3 of cutting.As a result, as shown in Fig. 2 D and table 2 below -1：
Table 2-1
It can be seen that, under sgRNA1 mediation, compared to SpCas9 nucleases (Cas9WT), this Cas9 nuclease of F916P When the DNA of mutant pair and sgRNA1 incomplementarities is cut, the ratio cut at the 4bp of PAM upstreams is significantly improved (U4) ratio, cut at the 5bp of PAM upstreams also significantly improves (U5), the ratio cut at the 3bp of PAM upstreams Reduce (U3).
As shown in Figure 2 E, using high throughput sequencing technologies detect DNA fragmentation delete jointing situation, except with expection Beyond the accurate connection (Joined precisely) being consistent, also exist compared with expected accurate connection, delete jointing Place adds " T " base, " AT " base, the situation of " CAT " base.During from different Cas9 nucleases, detect with it is pre- Accurate connection (Joined precisely) that phase is consistent, "+T " base, "+AT " base, the accounting of "+CAT " base are different.
Exemplified by selecting this Cas9 nuclease of F916P, accurate connection (Joined in line is detected Precisely accounting) is 11.07%, and DNA fragmentation, which is deleted, adds the accounting of " T " base for 27.18%, DNA at jointing The accounting that fragment deletes " AT " base added at jointing is 28.10%, and DNA fragmentation is deleted to be added at jointing The accounting of " CAT " base is 0.57%.
In view of DNA fragmentation is deleted detects the presence of the addition of " T " base (with expected accurate connection phase at jointing Than), it is presumed that DNA fragmentation, which deletes " T " base added at jointing, is derived from masterplate DNA (β-globin RE2 Point) on PAM (TGG) upstream 4bp at base.Also, further speculate Jie of this Cas9 nuclease of F916P in sgRNA2 Lead lower when being cut to genomic DNA fragment (β-globin RE2 sites), a pair DNA complementary with sgRNA is cut When, be to be cut at the 3bp of PAM upstreams, and pair with sgRNA incomplementarity DNAs cut when, then be on PAM (TGG) Swim and protruding terminus cutting is carried out at 4bp, so as to generate prominent broken ends D4.Prominent broken ends D4 is in cell repair system In the presence of produce DNA fragmentation delete when, result in DNA fragmentation delete jointing at " T " base addition.
Similarly, in view of the addition that " AT " base is detected the presence of at DNA fragmentation deletion jointing (precisely connects with expected Connect and compare), it is presumed that DNA fragmentation, which deletes " AT " base added at jointing, is derived from masterplate DNA (β-globin RE2 sites) on PAM (TGG) upstream 4bp and 5bp at base.Further speculate that this Cas9 nuclease of F916P exists When being cut under sgRNA2 mediation to genomic DNA fragment (β-globin RE2 sites), couple DNA complementary with sgRNA When chain is cut, cut at the 3bp of PAM upstreams, and pair with sgRNA incomplementarity DNAs cut when, be Protruding terminus cutting is carried out at PAM (TGG) upstream 5bp, so as to generate prominent broken ends D5.Prominent broken ends D5 is thin In the presence of born of the same parents' repair system produce DNA fragmentation delete when, result in DNA fragmentation delete jointing at " AT " base plus Enter.
Similarly, in view of DNA fragmentation delete jointing at detect the presence of " CAT " base addition (with it is expected precisely Connection compare), it is presumed that DNA fragmentation delete jointing at add " CAT " base be derived from masterplate DNA (β- Globin RE2 sites) on PAM (TGG) upstream 4bp, 5bp, 6bp at base.Further speculate this Cas9 core of F916P It is pair complementary with sgRNA when sour enzyme is cut under sgRNA2 mediation to genomic DNA fragment (β-globin RE2 sites) DNA when being cut, be to be cut at the 3bp of PAM upstreams, and when pair being cut with sgRNA incomplementarity DNAs, It is the progress protruding terminus cutting at PAM (TGG) upstream 6bp, so as to generate prominent broken ends D6.Prominent broken ends D5 When producing DNA fragmentation deletion in the presence of cell repair system, it result in DNA fragmentation and delete " CAT " base at jointing Addition.
And when F916P this Cas9 nuclease under sgRNA2 mediation to genomic DNA fragment (β-globin RE2 Point) when a pair DNA complementary with sgRNA is cut, cut at the 3bp of PAM upstreams when being cut, pair and It is the progress blunt ends cutting at PAM (TGG) upstream 3bp when sgRNA incomplementarity DNAs are cut, so as to generate blunt disconnected Split end D3.Blunt broken ends D3 will not cause DNA fragmentation when producing DNA fragmentation deletion in the presence of cell repair system The addition of base at jointing is deleted, but produces accurate connection (Joined precisely) in line.
It is therefore believed that：Under Cas9 nucleases F916P cutting, in the broken ends of generation, blunt broken ends D3 Accounting=11.07% of accurate connection (Joined precisely) that is consistent of accounting=expection.Prominent broken ends D4's Addition accounting=27.18% of accounting=" T " base.The addition accounting of prominent broken ends D5 accounting=" AT " base= 28.10%.Addition accounting=0.57% of prominent broken ends D6 accounting=" CAT " base.
But, it is observed that except accurate connection (Joined precisely) in line, DNA fragmentation are deleted In addition to " T " base, "+AT " base, "+CAT " base this four major classes situation is added at jointing, an also class is random Base deletes (Small deletion).It is considered that it is each fracture end that this kind of random base, which deletes (Small deletion), End (blunt broken ends D3/ protrudes broken ends D4/ and protrudes broken ends D5/ protrusions broken ends D6) is in cell repair system Randomly generated under effect, each broken ends are produced base with impartial probability and delete (Small deletion), each fracture Base of the end produced by the presence of cell repair system deletes the quantity and each broken ends of (Small deletion) Quantity be directly proportional.
The presence of phenomenon is deleted based on randomized bases, it is believed that, the actual measurement ratio of each broken ends obtained by sequencing There is gap, it is necessary to be modified reduction in example, i.e., with its actual proportions on the basis of the actual measurement ratio sum of various broken ends, The ratio of each broken ends is calculated, in this, as the accounting of the broken ends.I.e. to produced by Cas9 nucleases F916P cutting The accountings of each broken ends be standardized calculating, blunt broken ends D3 accounting is 16.55%
【Computational methods are：11.07% ÷ (11.07%+27.18%+28.10%+0.57%)】.
Prominent broken ends D4 ratio is 40.62%
【Computational methods are：27.18% ÷ (11.07%+27.18%+28.10%+0.57%)】.
Prominent broken ends D5 ratio is 41.98%
【Computational methods are：28.10% ÷ (11.07%+27.18%+28.10%+0.57%)】.
Prominent broken ends D6 ratio is 0.85%
【Computational methods are：0.57% ÷ (11.07%+27.18%+28.10%+0.57%)】.
That is, in the cutting mode that Cas9 nucleases F916P is carried out under sgRNA2 mediation to genomic DNA fragment, The accounting of D3 blunt ends cutting is that the accounting of 16.55%, D4 protruding terminuses cutting is accounting for for 40.62%, D5 protruding terminuses cutting Accounting than being cut for 41.98%, D6 protruding terminuses is 0.85%.
With reference to the above method, calculate wild type Cas9 nucleases and genomic DNA fragment is entered under sgRNA2 mediation In capable cutting mode, what accounting Y2, the D5 protruding terminus that accounting Y1, the D4 protruding terminus that D3 blunt ends are cut is cut was cut accounts for The accounting Y4 cut than Y3, D6 protruding terminus.As a result as shown in Fig. 2 F and table 2-2：
Table 2-2
It can be seen that, under sgRNA2 mediation, compared to SpCas9 nucleases (Cas9WT), F916P mutant is to genome When being cut in DNA fragmentation with the DNA of sgRNA2 incomplementarities, this mutant of F916P is cut at the 4bp of PAM upstreams The ratio cut is significantly improved (D4).
With reference to embodiment 1 method, according to Cas9 nucleases under sgRNA1 and sgRNA2 mediation respectively to genome DNA fragmentation carries out the ratio of cutting mode, predicts the sequence of the broken ends produced, and then extrapolates DNA fragmentation reversion downstream Base at jointing adds situation and ratio.As a result as shown in Figure 2 G, calculate that result is added with the base that experiment is detected Ratio is close.Cas9 nucleases are further confirmed under the mediation that sgRNA is combined, can be at the 3bp of PAM upstreams to farther Incomplementarity DNA is cut at base.
In addition, also by the Cas9 nucleic acid enzyme mutant Cas9-F916P of the present invention and control mutant K775A, R778A, E779A, K918P transfect human embryo kidney (HEK) HEK293T cells together with two sgRNAs for STM sites (β-globin RE1), Transfection collects genomic DNA after 48 hours, and pcr amplified DNA fragment deletion, reversion and again are carried out using high-flux sequence primer It is multiple, build storehouse and carry out high-flux sequence.The jointing base deleted and repeated according to DNA fragmentation adds situation, calculates these and dashes forward Cutting mode ratio of the variant under two sgRNAs mediations.
Sequence is targetted for the sgRNAs of STM sites (β-globin RE1)：
β-globin RE1sgRNA1：GATTGTTGTTGCCTTGGAGTG(SEQ ID NO.19)；
β-globin RE1sgRNA2：GCTGGTCCCCTGGTAACCTGG(SEQ ID NO.20)；
Forward and reverse deoxy-oligonucleotide：
β-globin RE1sgRNA1F：accgATTGTTGTTGCCTTGGAGTG(SEQ ID NO.21)；
β-globin RE1sgRNA1R：aaacCACTCCAAGGCAACAACAAT(SEQ ID NO.22)；
β-globin RE1sgRNA2F：accgCTGGTCCCCTGGTAACCTGG(SEQ ID NO.23)；
β-globin RE1sgRNA2R：aaacCCAGGTTACCAGGGGACCAG(SEQ ID NO.24)；
High flux primer：
Hiseq-hSTM-aF1:
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGCTTAGAGCCAGGACTAA TTGC；(SEQ ID NO.25)
Hiseq-hSTM-aR2:
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGGTGTAGAAATGAGCAA ATAAGT(SEQ ID NO.26)
Hiseq-hSTM-2F:
CAAGCAGAAGACGGCATACGAGATGATCGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGATTGAGTTCTG TTTGTTTCATCTAC(SEQ ID NO.27)
Hiseq-hSTM-2R:
CAAGCAGAAGACGGCATACGAGATAGTCAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAGCTCTGCCTGA AAGGAGTC(SEQ ID NO.28)。
As shown in figs.3 a and 3b, compared with wild type SpCas9 nucleases (abbreviation Cas9WT), control mutant K775A, The mode that R778A, E779A and K918P are cut under sgRNA1 and sgRNA2 mediation to genomic DNA fragment is not bright Aobvious change；And Cas9 nucleic acid enzyme mutant Cas9-F916P are compared with wild type SpCas9 nucleases (abbreviation Cas9WT), The mode cut under sgRNA1 and sgRNA2 mediation to genomic DNA fragment there occurs obvious change.
In summary, Cas9 nucleases (Cas9-F916P) of the invention are compared with wild type Cas9 nucleases, to purpose The protrusion broken ends and the ratio of blunt broken ends produced when genomic DNA fragment is cut are different.Using the present invention's Cas9 nucleases (Cas9-F916P) can be realized to the cutting to target gene group DNA fragmentation ad-hoc location and produced prominent disconnected End is split, the base complementary with prominent broken ends can be added in the way of filling-in is connected, and then the essence of ad-hoc location can be realized Quasi- DNA fragmentation editor.
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It is described above, only presently preferred embodiments of the present invention, it is not any to the present invention in form and substantial limitation, It should be pointed out that for those skilled in the art, on the premise of the inventive method is not departed from, can also make Some improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art, Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more Dynamic, modification and the equivalent variations developed, are the equivalent embodiment of the present invention；Meanwhile, all substantial technologicals pair according to the present invention The variation, modification and evolution for any equivalent variations that above-described embodiment is made, still fall within the scope of technical scheme It is interior.
SEQUENCE LISTING
<110>Shanghai Communications University
<120>A kind of Cas9 nucleases F916P and application thereof
<130> 171286
<160> 28
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Artificial
<220>
<223> HS51 RE1sgRNA1
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gccacacatc caaggctgac 20
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gagatttggg gcgtcaggaa g 21
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<223> Hiseq-hHs51-aF
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atgatacggc gaccaccgag atctacactc tttccctaca cgacgctctt ccgatctgca 60
aggagatccg tgtcgtc 77
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aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctaa 60
ggatgttgtg gaaggcgagc ag 82
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caagcagaag acggcatacg agatggacgg gtgactggag ttcagacgtg tgctcttccg 60
atctctttac atgacagctt ccggtag 87
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caagcagaag acggcatacg agatttgact gtgactggag ttcagacgtg tgctcttccg 60
atcttttttg gctaacaaca tagtgcttc 89
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Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser
20 25 30
Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys
35 40 45
Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu
50 55 60
Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg
65 70 75 80
Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile
85 90 95
Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp
100 105 110
Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys
115 120 125
Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala
130 135 140
Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val
145 150 155 160
Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala
165 170 175
His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn
180 185 190
Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr
195 200 205
Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp
210 215 220
Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu
225 230 235 240
Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly
245 250 255
Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn
260 265 270
Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr
275 280 285
Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala
290 295 300
Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser
305 310 315 320
Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala
325 330 335
Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu
340 345 350
Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe
355 360 365
Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala
370 375 380
Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met
385 390 395 400
Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu
405 410 415
Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His
420 425 430
Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro
435 440 445
Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg
450 455 460
Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala
465 470 475 480
Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu
485 490 495
Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met
500 505 510
Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His
515 520 525
Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val
530 535 540
Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu
545 550 555 560
Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val
565 570 575
Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe
580 585 590
Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu
595 600 605
Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu
610 615 620
Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu
625 630 635 640
Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr
645 650 655
Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg
660 665 670
Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg
675 680 685
Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
690 695 700
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr
705 710 715 720
Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser
725 730 735
Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys
740 745 750
Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met
755 760 765
Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn
770 775 780
Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg
785 790 795 800
Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His
805 810 815
Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr
820 825 830
Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn
835 840 845
Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu
850 855 860
Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn
865 870 875 880
Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met
885 890 895
Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg
900 905 910
Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu
915 920 925
Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
930 935 940
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr
945 950 955 960
Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys
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Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val
980 985 990
Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
995 1000 1005
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser
1010 1015 1020
Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1025 1030 1035
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr
1040 1045 1050
Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
1055 1060 1065
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1070 1075 1080
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1085 1090 1095
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1100 1105 1110
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu
1115 1120 1125
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp
1130 1135 1140
Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
1145 1150 1155
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys
1160 1165 1170
Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
1175 1180 1185
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1190 1195 1200
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1205 1210 1215
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1220 1225 1230
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1235 1240 1245
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys
1250 1255 1260
Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1265 1270 1275
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe
1280 1285 1290
Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
1295 1300 1305
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1310 1315 1320
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1325 1330 1335
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
1340 1345 1350
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
1355 1360 1365
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
1370 1375 1380
Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys
1385 1390 1395
Lys Lys Lys
1400
<210> 8
<211> 4206
<212> DNA
<213> Artificial
<220>
<223> SpCas9
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atggccccaa agaagaagcg gaaggtcggt atccacggtg tcccagcagc catggacaag 60
aagtactcca ttgggctcga tatcggcaca aacagcgtcg gctgggccgt cattacggac 120
gagtacaagg tgccgagcaa aaaattcaaa gttctgggca ataccgatcg ccacagcata 180
aagaagaacc tcattggcgc cctcctgttc gactccgggg agacggccga agccacgcgg 240
ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga atcggatctg ctacctgcag 300
gagatcttta gtaatgagat ggctaaggtg gatgactctt tcttccatag gctggaggag 360
tcctttttgg tggaggagga taaaaagcac gagcgccacc caatctttgg caatatcgtg 420
gacgaggtgg cgtaccatga aaagtaccca accatatatc atctgaggaa gaagcttgta 480
gacagtactg ataaggctga cttgcggttg atctatctcg cgctggcgca tatgatcaaa 540
tttcggggac acttcctcat cgagggggac ctgaacccag acaacagcga tgtcgacaaa 600
ctctttatcc aactggttca gacttacaat cagcttttcg aagagaaccc gatcaacgca 660
tccggagttg acgccaaagc aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa 720
aacctcatcg cacagctccc tggggagaag aagaacggcc tgtttggtaa tcttatcgcc 780
ctgtcactcg ggctgacccc caactttaaa tctaacttcg acctggccga agatgccaag 840
cttcaactga gcaaagacac ctacgatgat gatctcgaca atctgctggc ccagatcggc 900
gaccagtacg cagacctttt tttggcggca aagaacctgt cagacgccat tctgctgagt 960
gatattctgc gagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag 1020
cgctatgatg agcaccacca agacttgact ttgctgaagg cccttgtcag acagcaactg 1080
cctgagaagt acaaggaaat tttcttcgat cagtctaaaa atggctacgc cggatacatt 1140
gacggcggag caagccagga ggaattttac aaatttatta agcccatctt ggaaaaaatg 1200
gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcg caaacagcgc 1260
actttcgaca atggaagcat cccccaccag attcacctgg gcgaactgca cgctatactc 1320
aggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc 1380
ctcacatttc ggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg 1440
tggatgactc gcaaatcaga agagaccatc actccctgga acttcgagga agtcgtggat 1500
aagggggcct ctgcccagtc cttcatcgaa aggatgacta actttgataa aaatctgcct 1560
aacgaaaagg tgcttcctaa acactctctg ctgtacgagt acttcacagt ttataacgag 1620
ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcct gtctggagag 1680
cagaagaaag ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacag 1740
ctcaaagaag actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg 1800
gaggatcgct tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat cattaaagac 1860
aaggacttcc tggacaatga ggagaacgag gacattcttg aggacattgt cctcaccctt 1920
acgttgtttg aagataggga gatgattgaa gaacgcttga aaacttacgc tcatctcttc 1980
gacgacaaag tcatgaaaca gctcaagagg cgccgatata caggatgggg gcggctgtca 2040
agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcct ggattttctt 2100
aagtccgatg gatttgccaa ccggaacttc atgcagttga tccatgatga ctctctcacc 2160
tttaaggagg acatccagaa agcacaagtt tctggccagg gggacagtct tcacgagcac 2220
atcgctaatc ttgcaggtag cccagctatc aaaaagggaa tactgcagac cgttaaggtc 2280
gtggatgaac tcgtcaaagt aatgggaagg cataagcccg agaatatcgt tatcgagatg 2340
gcccgagaga accaaactac ccagaaggga cagaagaaca gtagggaaag gatgaagagg 2400
attgaagagg gtataaaaga actggggtcc caaatcctta aggaacaccc agttgaaaac 2460
acccagcttc agaatgagaa gctctacctg tactacctgc agaacggcag ggacatgtac 2520
gtggatcagg aactggacat caatcggctc tccgactacg acgtggatca tatcgtgccc 2580
cagtcttttc tcaaagatga ttctattgat aataaagtgt tgacaagatc cgataaaaat 2640
agagggaaga gtgataacgt cccctcagaa gaagttgtca agaaaatgaa aaattattgg 2700
cggcagctgc tgaacgccaa actgatcaca caacggaagt tcgataatct gactaaggct 2760
gaacgaggtg gcctgtctga gttggataaa gcaggcttca tcaaaaggca gcttgttgag 2820
acacgccaga tcaccaagca cgtggcccaa attctcgatt cacgcatgaa caccaagtac 2880
gatgaaaatg acaaactgat tcgagaggtg aaagttatta ctctgaagtc taagctggtc 2940
tcagatttca gaaaggactt tcagttttat aaggtgagag agatcaacaa ttaccaccat 3000
gcgcatgatg cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag 3060
cttgaatctg aatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca 3120
aagtctgagc aggaaatagg caaggccacc gctaagtact tcttttacag caatattatg 3180
aattttttca agaccgagat tacactggcc aatggagaga ttcggaagcg accacttatc 3240
gaaacaaacg gagaaacagg agaaatcgtg tgggacaagg gtagggattt cgcgacagtc 3300
cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt acagaccgga 3360
ggcttctcca aggaaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa 3420
aaagattggg accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta 3480
ctggttgtgg ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg 3540
ctgggcatca caatcatgga gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag 3600
gcgaaaggat ataaagaggt caaaaaagac ctcatcatta agcttcccaa gtactctctc 3660
tttgagcttg aaaacggccg gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt 3720
aacgagctgg cactgccctc taaatacgtt aatttcttgt atctggccag ccactatgaa 3780
aagctcaaag ggtctcccga agataatgag cagaagcagc tgttcgtgga acaacacaaa 3840
cactaccttg atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc 3900
gacgctaacc tcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg 3960
gagcaggcag aaaacattat ccacttgttt actctgacca acttgggcgc gcctgcagcc 4020
ttcaagtact tcgacaccac catagacaga aagcggtaca cctctacaaa ggaggtcctg 4080
gacgccacac tgattcatca gtcaattacg gggctctatg aaacaagaat cgacctctct 4140
cagctcggtg gagacaagcg tcctgctgct actaagaaag ctggtcaagc taagaaaaag 4200
aaataa 4206
<210> 9
<211> 1401
<212> PRT
<213> Artificial
<220>
<223> Cas9-F916P
<400> 9
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser
20 25 30
Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys
35 40 45
Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu
50 55 60
Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg
65 70 75 80
Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile
85 90 95
Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp
100 105 110
Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys
115 120 125
Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala
130 135 140
Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val
145 150 155 160
Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala
165 170 175
His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn
180 185 190
Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr
195 200 205
Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp
210 215 220
Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu
225 230 235 240
Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly
245 250 255
Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn
260 265 270
Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr
275 280 285
Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala
290 295 300
Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser
305 310 315 320
Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala
325 330 335
Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu
340 345 350
Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe
355 360 365
Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala
370 375 380
Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met
385 390 395 400
Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu
405 410 415
Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His
420 425 430
Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro
435 440 445
Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg
450 455 460
Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala
465 470 475 480
Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu
485 490 495
Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met
500 505 510
Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His
515 520 525
Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val
530 535 540
Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu
545 550 555 560
Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val
565 570 575
Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe
580 585 590
Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu
595 600 605
Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu
610 615 620
Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu
625 630 635 640
Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr
645 650 655
Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg
660 665 670
Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg
675 680 685
Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
690 695 700
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr
705 710 715 720
Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser
725 730 735
Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys
740 745 750
Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met
755 760 765
Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn
770 775 780
Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg
785 790 795 800
Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His
805 810 815
Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr
820 825 830
Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn
835 840 845
Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu
850 855 860
Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn
865 870 875 880
Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met
885 890 895
Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg
900 905 910
Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu
915 920 925
Asp Lys Ala Gly Pro Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
930 935 940
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr
945 950 955 960
Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys
965 970 975
Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val
980 985 990
Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
995 1000 1005
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser
1010 1015 1020
Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1025 1030 1035
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr
1040 1045 1050
Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
1055 1060 1065
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1070 1075 1080
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1085 1090 1095
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1100 1105 1110
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu
1115 1120 1125
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp
1130 1135 1140
Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
1145 1150 1155
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys
1160 1165 1170
Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
1175 1180 1185
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1190 1195 1200
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1205 1210 1215
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1220 1225 1230
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1235 1240 1245
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys
1250 1255 1260
Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1265 1270 1275
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe
1280 1285 1290
Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
1295 1300 1305
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1310 1315 1320
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1325 1330 1335
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
1340 1345 1350
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
1355 1360 1365
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
1370 1375 1380
Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys
1385 1390 1395
Lys Lys Lys
1400
<210> 10
<211> 4206
<212> DNA
<213> Artificial
<220>
<223> Cas9-F916P
<400> 10
atggccccaa agaagaagcg gaaggtcggt atccacggtg tcccagcagc catggacaag 60
aagtactcca ttgggctcga tatcggcaca aacagcgtcg gctgggccgt cattacggac 120
gagtacaagg tgccgagcaa aaaattcaaa gttctgggca ataccgatcg ccacagcata 180
aagaagaacc tcattggcgc cctcctgttc gactccgggg agacggccga agccacgcgg 240
ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga atcggatctg ctacctgcag 300
gagatcttta gtaatgagat ggctaaggtg gatgactctt tcttccatag gctggaggag 360
tcctttttgg tggaggagga taaaaagcac gagcgccacc caatctttgg caatatcgtg 420
gacgaggtgg cgtaccatga aaagtaccca accatatatc atctgaggaa gaagcttgta 480
gacagtactg ataaggctga cttgcggttg atctatctcg cgctggcgca tatgatcaaa 540
tttcggggac acttcctcat cgagggggac ctgaacccag acaacagcga tgtcgacaaa 600
ctctttatcc aactggttca gacttacaat cagcttttcg aagagaaccc gatcaacgca 660
tccggagttg acgccaaagc aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa 720
aacctcatcg cacagctccc tggggagaag aagaacggcc tgtttggtaa tcttatcgcc 780
ctgtcactcg ggctgacccc caactttaaa tctaacttcg acctggccga agatgccaag 840
cttcaactga gcaaagacac ctacgatgat gatctcgaca atctgctggc ccagatcggc 900
gaccagtacg cagacctttt tttggcggca aagaacctgt cagacgccat tctgctgagt 960
gatattctgc gagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag 1020
cgctatgatg agcaccacca agacttgact ttgctgaagg cccttgtcag acagcaactg 1080
cctgagaagt acaaggaaat tttcttcgat cagtctaaaa atggctacgc cggatacatt 1140
gacggcggag caagccagga ggaattttac aaatttatta agcccatctt ggaaaaaatg 1200
gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcg caaacagcgc 1260
actttcgaca atggaagcat cccccaccag attcacctgg gcgaactgca cgctatactc 1320
aggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc 1380
ctcacatttc ggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg 1440
tggatgactc gcaaatcaga agagaccatc actccctgga acttcgagga agtcgtggat 1500
aagggggcct ctgcccagtc cttcatcgaa aggatgacta actttgataa aaatctgcct 1560
aacgaaaagg tgcttcctaa acactctctg ctgtacgagt acttcacagt ttataacgag 1620
ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcct gtctggagag 1680
cagaagaaag ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacag 1740
ctcaaagaag actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg 1800
gaggatcgct tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat cattaaagac 1860
aaggacttcc tggacaatga ggagaacgag gacattcttg aggacattgt cctcaccctt 1920
acgttgtttg aagataggga gatgattgaa gaacgcttga aaacttacgc tcatctcttc 1980
gacgacaaag tcatgaaaca gctcaagagg cgccgatata caggatgggg gcggctgtca 2040
agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcct ggattttctt 2100
aagtccgatg gatttgccaa ccggaacttc atgcagttga tccatgatga ctctctcacc 2160
tttaaggagg acatccagaa agcacaagtt tctggccagg gggacagtct tcacgagcac 2220
atcgctaatc ttgcaggtag cccagctatc aaaaagggaa tactgcagac cgttaaggtc 2280
gtggatgaac tcgtcaaagt aatgggaagg cataagcccg agaatatcgt tatcgagatg 2340
gcccgagaga accaaactac ccagaaggga cagaagaaca gtagggaaag gatgaagagg 2400
attgaagagg gtataaaaga actggggtcc caaatcctta aggaacaccc agttgaaaac 2460
acccagcttc agaatgagaa gctctacctg tactacctgc agaacggcag ggacatgtac 2520
gtggatcagg aactggacat caatcggctc tccgactacg acgtggatca tatcgtgccc 2580
cagtcttttc tcaaagatga ttctattgat aataaagtgt tgacaagatc cgataaaaat 2640
agagggaaga gtgataacgt cccctcagaa gaagttgtca agaaaatgaa aaattattgg 2700
cggcagctgc tgaacgccaa actgatcaca caacggaagt tcgataatct gactaaggct 2760
gaacgaggtg gcctgtctga gttggataaa gcaggcccca tcaaaaggca gcttgttgag 2820
acacgccaga tcaccaagca cgtggcccaa attctcgatt cacgcatgaa caccaagtac 2880
gatgaaaatg acaaactgat tcgagaggtg aaagttatta ctctgaagtc taagctggtc 2940
tcagatttca gaaaggactt tcagttttat aaggtgagag agatcaacaa ttaccaccat 3000
gcgcatgatg cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag 3060
cttgaatctg aatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca 3120
aagtctgagc aggaaatagg caaggccacc gctaagtact tcttttacag caatattatg 3180
aattttttca agaccgagat tacactggcc aatggagaga ttcggaagcg accacttatc 3240
gaaacaaacg gagaaacagg agaaatcgtg tgggacaagg gtagggattt cgcgacagtc 3300
cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt acagaccgga 3360
ggcttctcca aggaaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa 3420
aaagattggg accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta 3480
ctggttgtgg ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg 3540
ctgggcatca caatcatgga gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag 3600
gcgaaaggat ataaagaggt caaaaaagac ctcatcatta agcttcccaa gtactctctc 3660
tttgagcttg aaaacggccg gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt 3720
aacgagctgg cactgccctc taaatacgtt aatttcttgt atctggccag ccactatgaa 3780
aagctcaaag ggtctcccga agataatgag cagaagcagc tgttcgtgga acaacacaaa 3840
cactaccttg atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc 3900
gacgctaacc tcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg 3960
gagcaggcag aaaacattat ccacttgttt actctgacca acttgggcgc gcctgcagcc 4020
ttcaagtact tcgacaccac catagacaga aagcggtaca cctctacaaa ggaggtcctg 4080
gacgccacac tgattcatca gtcaattacg gggctctatg aaacaagaat cgacctctct 4140
cagctcggtg gagacaagcg tcctgctgct actaagaaag ctggtcaagc taagaaaaag 4200
aaataa 4206
<210> 11
<211> 20
<212> DNA
<213> Artificial
<220>
<223> β-globin RE2sgRNA1
<400> 11
acccaatgac ctcaggctgt 20
<210> 12
<211> 20
<212> DNA
<213> Artificial
<220>
<223> β-globin RE2sgRNA2
<400> 12
tcacttgtta gcggcatctg 20
<210> 13
<211> 25
<212> DNA
<213> Artificial
<220>
<223> Cas9-F916P-F
<400> 13
taaagcaggc cccatcaaaa ggcag 25
<210> 14
<211> 18
<212> DNA
<213> Artificial
<220>
<223> Cas9-F916P-R
<400> 14
tccaactcag acaggcca 18
<210> 15
<211> 86
<212> DNA
<213> Artificial
<220>
<223> Hiseq-RRM-1F3
<400> 15
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctat 60
atggcatcct agccttaaga aactag 86
<210> 16
<211> 81
<212> DNA
<213> Artificial
<220>
<223> Hiseq-RRM-1R2
<400> 16
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctta 60
cgacgcagga gccgtatcat g 81
<210> 17
<211> 89
<212> DNA
<213> Artificial
<220>
<223> Hiseq-RRM-3F2
<400> 17
caagcagaag acggcatacg agataagcta gtgactggag ttcagacgtg tgctcttccg 60
atctatagca atgaaatctt gaaggagtg 89
<210> 18
<211> 85
<212> DNA
<213> Artificial
<220>
<223> Hiseq-RRM-3R2
<400> 18
caagcagaag acggcatacg agattcaagt gtgactggag ttcagacgtg tgctcttccg 60
atctgcacag ccctgctcta ttacg 85
<210> 19
<211> 21
<212> DNA
<213> Artificial
<220>
<223> β-globin RE1sgRNA1
<400> 19
gattgttgtt gccttggagt g 21
<210> 20
<211> 21
<212> DNA
<213> Artificial
<220>
<223> β-globin RE1sgRNA2
<400> 20
gctggtcccc tggtaacctg g 21
<210> 21
<211> 24
<212> DNA
<213> Artificial
<220>
<223> β-globin RE1sgRNA1F
<400> 21
accgattgtt gttgccttgg agtg 24
<210> 22
<211> 24
<212> DNA
<213> Artificial
<220>
<223> β-globin RE1sgRNA1R
<400> 22
aaaccactcc aaggcaacaa caat 24
<210> 23
<211> 24
<212> DNA
<213> Artificial
<220>
<223> β-globin RE1sgRNA2F
<400> 23
accgctggtc ccctggtaac ctgg 24
<210> 24
<211> 24
<212> DNA
<213> Artificial
<220>
<223> β-globin RE1sgRNA2R
<400> 24
aaacccaggt taccagggga ccag 24
<210> 25
<211> 81
<212> DNA
<213> Artificial
<220>
<223> Hiseq-hSTM-aF1
<400> 25
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttg 60
cttagagcca ggactaattg c 81
<210> 26
<211> 83
<212> DNA
<213> Artificial
<220>
<223> Hiseq-hSTM-aR2
<400> 26
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttg 60
ggtgtagaaa tgagcaaata agt 83
<210> 27
<211> 91
<212> DNA
<213> Artificial
<220>
<223> Hiseq-hSTM-2F
<400> 27
caagcagaag acggcatacg agatgatcgt gtgactggag ttcagacgtg tgctcttccg 60
atctagattg agttctgttt gtttcatcta c 91
<210> 28
<211> 85
<212> DNA
<213> Artificial
<220>
<223> Hiseq-hSTM-2R
<400> 28
caagcagaag acggcatacg agatagtcaa gtgactggag ttcagacgtg tgctcttccg 60
atctcagctc tgcctgaaag gagtc 85

Claims (20)

1. a kind of Cas9 nucleases, with Cas9 nucleases, it is adaptable to CRISPR/Cas9 systems, the Cas9 nucleases It is that the amino acid of wild type Cas9 nucleases the 916th is mutated into Pro by Phe to obtain.
2. Cas9 nucleases according to claim 1, it is characterised in that the Cas9 nucleases and wild type Cas9 nucleic acid Enzyme is compared, and the protrusion broken ends and the ratio of blunt broken ends produced when being cut to target gene group DNA fragmentation are different.
3. Cas9 nucleases according to claim 1, it is characterised in that the wild type Cas9 nucleases are SpCas9.
4. Cas9 nucleases according to claim 3, it is characterised in that the amino acid sequence of the wild type Cas9 nucleases Row are as shown in SEQ ID NO.7.
5. Cas9 nucleases according to claim 1, it is characterised in that the Cas9 nucleases contain such as SEQ ID Amino acid sequence shown in NO.9.
6. a kind of polynucleotides, it encodes the Cas9 nucleases as described in any one of Claims 1 to 5.
7. a kind of expression vector, it contains polynucleotides as claimed in claim 6.
8. a kind of host cell, it is converted by expression vector as claimed in claim 7.
9. a kind of method of the Cas9 nucleases prepared as described in any one of Claims 1 to 5, including step：Build containing such as The expression vector of any one of Claims 1 to 5 Cas9 nucleases encoding polynucleotide sequence, then by the expression vector Conversion induced expression into host cell, separation obtains described Cas9 nucleases from expression product.
10. Cas9 nucleases or polynucleotides as claimed in claim 6 or such as power as described in any one of Claims 1 to 5 Profit requires that the expression vector described in 7 is used for genomic DNA fragment editor or the use for preparing genomic DNA fragment edit tool On the way.
11. purposes according to claim 10, it is characterised in that the editor includes unit point editor and many sites are compiled Volume, the edit bit points of many sites editor for two and more than.
12. purposes according to claim 10, it is characterised in that the mode of the editor include mutation, delete, reversion or Inversion, repetition, transposition or insertion.
13. a kind of DNA genomic fragments edit tool, is CRISPR/Cas9 systems, the CRISPR/Cas9 systems are included such as Cas9 nucleases described in any one of Claims 1 to 5 or polynucleotides as claimed in claim 6 or such as claim 7 institute The expression vector stated.
14. a kind of DNA fragmentation edit tool according to claim 13, it is characterised in that the CRISPR/Cas9 systems Also include for target DNA fragment one or more sgRNA, it is the multiple refer to two and more than.
15. a kind of genomic DNA fragment edit methods, using the Cas9 nucleases as described in any one of Claims 1 to 4 and with Cooperation one or more sgRNA, edlin is entered to genomic DNA fragment to be edited using CRISPR/Cas9 systems.
16. genomic DNA fragment edit methods according to claim 15, it is characterised in that the editor includes unit Point editor and many sites editor, the edit bit points of many sites editor for two and more than.
17. genomic DNA fragment edit methods according to claim 15, the mode of the editor include mutation, delete, Reversion or inversion, repetition, transposition or insertion.
18. genomic DNA fragment edit methods according to claim 15, it is characterised in that will be as claimed in claim 6 The expression vector containing Cas9 nuclease coded polynucleotides and matched one or more sgRNA be together transferred to carefully In born of the same parents, edlin is entered to genomic DNA fragment to be edited.
19. a kind of genomic DNA fragment unit point edit methods, using CRISPR/Cas9 systems, using such as Claims 1 to 5 Cas9 nucleases described in any one carry out cutting to DNA double chain and produce prominent broken ends, by cell itself repair system, The base complementary with prominent broken ends is added in the way of filling-in is connected.
20. genomic DNA fragment unit point edit methods according to claim 19, it is characterised in that methods described can The feature of base mutation during changing unit point editor.
CN201710344461.4A 2017-05-16 2017-05-16 A kind of Cas9 nucleases F916P and application thereof Pending CN106987571A (en)

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WO2023212657A3 (en) * 2022-04-27 2023-12-07 New York University Enhancement of safety and precision for crispr-cas induced gene editing by variants of dna polymerase using cas-plus variants

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WO2016196655A1 (en) * 2015-06-03 2016-12-08 The Regents Of The University Of California Cas9 variants and methods of use thereof

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WO2016196655A1 (en) * 2015-06-03 2016-12-08 The Regents Of The University Of California Cas9 variants and methods of use thereof

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023212657A3 (en) * 2022-04-27 2023-12-07 New York University Enhancement of safety and precision for crispr-cas induced gene editing by variants of dna polymerase using cas-plus variants

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