WO2021065155A1 - METHOD FOR CONTROLLING DNA CLEAVAGE ACTIVITY OF Cas9 NUCLEASE - Google Patents

Abstract

Provided is a method for controlling the DNA cleavage activity of Cas9 nuclease, the method comprising (1) a step for bringing a CRISPR-Cas9 ribonucleoprotein complex into contact with a target DNA, wherein the complex comprises a guide RNA and an inactivated Cas9 nuclease containing a point mutation at the histidine residue corresponding to the position 840 according to numbering based on the amino acid sequence of a Streptococcus pyogenes-derived Cas9 nuclease; and then (2) a step for bringing the complex into contact with imidazole or a derivative thereof.

Description

Method of controlling DNA cleavage activity of Cas9 nuclease

The present invention relates to a method for controlling the DNA cleavage activity of Cas9 nuclease.

The CRISPR-Cas9 system is currently the most noticeable genome editing technology. The Cas9 nuclease forms a complex with the guide RNA and specifically cleaves any DNA sequence targeted by the guide RNA. However, the CRISPR-Cas9 system has an off-target effect that non-specifically cleaves similar sequences other than the target sequence, and it is necessary to suppress the off-target effect in order to improve the accuracy of genome editing. There is. The off-target effect is a method of temporally controlling the DNA cleavage activity of the activated CRISPR-Cas9 ribonucleoprotein (RNP) complex because the longer it is present in the cell, the greater the increase. Is desired.

Zetsche et al. Report a rapamycin-induced split Cas9 (Non-Patent Document 1). In this method, two split Cas9 fragments fused with FKBP and FRB are used, and when Cas9 is reconstituted by the addition of rapamycin, the DNA cleavage activity of Cas9 is induced. This method is the first report to control the initiation of genome editing at the protein level. However, due to the high affinity of rapamycin, FKBP, and FRB, removal of rapamycin retains the reconstituted Cas9 and cannot stop genome editing, thus controlling genome editing in time. Can't. In addition, Nihongaki et al. Have reported a method of inducing the DNA cleavage activity of Cas9 by irradiating with light using two split Cas9 fragments in which photoinducible dimerization domains are fused with each other (non-patent). Document 2). However, even this method has not succeeded in rapidly stopping the DNA-cleaving activity of the reconstituted Cas9.

Zetsche, B. et al., Nat. Biotechnol., Vol. 33, pp. 139-142 (2015) Nihongaki, Y. et al., Nat. Biotechnol., Vol. 33, pp. 755-760 (2015)

The present invention has been made for the purpose of solving various problems of the prior art and providing a CRISPR-Cas9 system capable of reversibly turning on / off the DNA cleavage activity.

As a result of diligent research, the inventors have found that the DNA cleavage activity of Cas9 can be controlled in time by adopting a "chemical rescue" method in which the function of the protein reduced by mutation is restored by the presence of a low molecular weight compound. I found it.

That is, according to one embodiment, the present invention is a method for controlling the DNA cleavage activity of Cas9 nuclease, which includes (1) a step of contacting a CRISPR-Cas9 ribonuclear protein complex with a target DNA, and here. The complex comprises a guide RNA and an inactivated Cas9 nuclease containing a point mutation at the histidine residue corresponding to position 840 in the numbering based on the amino acid sequence of Cas9 nuclease derived from pyogenic Cas9 nuclease, followed by (2) The present invention provides a method including a step of contacting the complex with imidazole or a derivative thereof.

The above method preferably further includes (3) after the step (2), a step of separating imidazole or a derivative thereof from the complex.

The point mutation is preferably a substitution with an alanine residue or a glycine residue.

The imidazole or its derivative preferably has a concentration of 1 to 500 mM.

The imidazole or a derivative thereof is preferably selected from the group consisting of imidazole and an imidazole derivative having a substituent at the 2-position and / or 4-position of the imidazole ring.

The above method may be performed in vitro or in vivo.

In addition, according to one embodiment, the present invention is an inactivated Cas9 nuclease containing a point mutation at the histidine residue corresponding to the 840th position in the numbering based on the amino acid sequence of Cas9 nuclease derived from Streptococcus pyogenes or the like. Provided is a kit for genome editing containing a nucleic acid encoding imidazole or a derivative thereof.

According to the method according to the present invention, the DNA cleavage activity of Cas9 can be controlled in time. Therefore, according to the method according to the present invention, the off-target effect of the CRISPR-Cas9 system can be minimized, and genome editing with high accuracy becomes possible.

FIG. 1A is a schematic diagram of a CRISPR-Cas9 complex containing wild-type Cas9. FIG. 1B is a schematic diagram of a CRISPR-Cas9 complex containing the Cas9 mutant H840A. FIG. 1C is a schematic diagram of the chemical rescue of the DNA cleavage activity of Cas9 mutant H840A by imidazole. FIG. 2 is a diagram showing the DNA cleavage activity of Cas9 mutants H840A and H840G in the absence or presence of imidazole. FIG. 3 is a diagram showing the DNA cleavage activity of Cas9 mutants H983A and H983G in the absence or presence of imidazole. FIG. 4 is a diagram showing the DNA cleavage activity of Cas9 mutants H840A and H840G at various imidazole concentrations. FIG. 5 is a diagram showing the recovery of the DNA cleavage activity of Cas9 mutant H840A over time in the presence of imidazole. FIG. 6 is a diagram showing the recovery of the DNA cleavage activity of Cas9 mutant H840G over time in the presence of imidazole. FIG. 7 is a diagram showing the restoration of the DNA cleavage activity of Cas9 mutant H840A by imidazole at various magnesium chloride concentrations. FIG. 8 is a diagram showing the restoration of the DNA cleavage activity of Cas9 mutant H840G by imidazole at various magnesium chloride concentrations. FIG. 9 is a diagram showing the recovery of the DNA cleavage activity of Cas9 mutant H840A by various imidazole derivatives. FIG. 10 is a diagram showing the restoration of the DNA cleavage activity of Cas9 mutant H840G by various imidazole derivatives.

Hereinafter, the present invention will be described in detail, but the present invention is not limited to the embodiments described in the present specification.

According to the first embodiment, the present invention is a method for controlling the DNA cleavage activity of Cas9 nuclease, which includes (1) contacting the target DNA with the CRISPR-Cas9 ribonuclear protein complex, and here. The complex comprises a guide RNA and an inactivated Cas9 nuclease containing a point mutation at the histidine residue corresponding to position 840 in the numbering based on the amino acid sequence of Cas9 nuclease derived from pyogenic Cas9 nuclease, followed by (2) A method comprising contacting the complex with imidazole or a derivative thereof.

The method of this embodiment uses a CRISPR-Cas9 ribonuclear protein complex containing a guide RNA and an inactivated Cas9 nuclease. The "CRISPR-Cas9 ribonuclear protein complex" means a complex in which Cas9 nuclease is induced site-specifically recognized by a guide RNA and can interact with a target DNA sequence. Hereinafter, in the present specification, the CRISPR-Cas9 ribonuclear protein complex containing a guide RNA and an inactivated Cas9 nuclease will also be referred to as an "inactive CRISPR-Cas9 complex".

The "guide RNA" means an RNA that contains a guide sequence complementary to the target DNA sequence and has a function of guiding the CRISPR-Cas9 ribonuclear protein complex to the target DNA and specifically binding to the target DNA. The structure of the guide RNA is not particularly limited as long as it contains the guide sequence as long as it has the above-mentioned function in the CRISPR-Cas9 system. That is, the guide RNA in the present embodiment may be a dual RNA of crRNA and trans-activating crRNA (tracrRNA), or a single-strand guide RNA (sgRNA) in which crRNA and tracrRNA are linked by a linker. You may. A method for designing a guide RNA has already been established, and for example, it can be designed using a design tool known in the art (for example, CRISPR (http://crispr.dbcls.jp)).

The "inactivated Cas9 nuclease" in this embodiment includes a point mutation in the histidine residue corresponding to position 840 in the numbering based on the amino acid sequence of Cas9 nuclease derived from Streptococcus pyogenes.

The "Cas9 nuclease" in the present embodiment is derived from any bacterium such as Streptococcus pneumoniae, Streptococcus thermophilus, and Cas9 nuclease, which may be Cas9 nuclease. , Preferably Cas9 nuclease derived from Streptococcus pyogenes (SEQ ID NO: 12; NCBI Reference Sequence (RefSeq) ID: WP_010922251).

In addition, the Cas9 nuclease in this embodiment maintains the same physiological function as the Cas9 nuclease derived from bacteria (that is, it is activated when the CRISPR-Cas9 ribonuclear protein complex is constructed, and the target DNA sequence is set. These variants, homologues, etc. may be included as long as they are cleaved). Therefore, the Cas9 nuclease in this embodiment maintains the same physiological function as the Cas9 nuclease derived from bacteria (that is, it is activated when the CRISPR-Cas9 ribonuclear protein complex is constructed, and the target DNA sequence is set. A protein consisting of an amino acid sequence having 80% or more, preferably 90% or more, more preferably about 95% or more identity with the Cas9 nuclease derived from the above bacteria can be included. Amino acid sequence identity can be calculated using sequence analysis software or using programs commonly used in the art (FASTA, BLAST, etc.). Sequence information of Cas9 nuclease derived from bacteria can be obtained from a predetermined database or the like.

The inactivated Cas9 nuclease in the present embodiment is the 840th position in the Cas9 nuclease defined above by any conventionally known genetic engineering method, numbered based on the amino acid sequence of the Cas9 nuclease derived from Streptococcus pyogenes. It can be prepared by introducing a point mutation into the histidine residue corresponding to. In the inactivated Cas9 nuclease in the present embodiment, the amino acid residue introduced in place of the histidine residue by a point mutation may be arbitrary, but is preferably a non-polar amino acid residue, and is preferably an alanine residue or glycine. It is particularly preferably a residue.

In the method of this embodiment, the target DNA is contacted with the inactive CRISPR-Cas9 complex. The method of this embodiment may be carried out in vitro (ie, in a reaction solution) or in vivo (ie, intracellularly).

When the method of the present embodiment is carried out in vitro, for example, a reaction solution containing the target DNA and the inactive CRISPR-Cas9 complex may be prepared, and the reaction solution may be incubated for a certain period of time. The composition of the reaction solution is not particularly limited as long as it is suitable for the enzymatic activity of Cas9 nuclease, and is appropriately determined according to the composition of the reaction solution used in the already established CRISPR-Cas9 genome editing method. can do. For example, the reaction solution is prepared by buffering the inactive CRISPR-Cas9 complex with the target DNA (for example, 1 to 100 mM HEPES (pH 7.0 to 8.0)) and 1 to 100 mM Tris (pH 7.0 to 8). It can be prepared by adding to an aqueous solvent containing (eg. 0)) and / or a salt (eg, 50-300 mM NaCl, 50-300 mM KCl, 0-100 mM MgCl _{2, etc.).} The final concentration of the Inactive CRISPR-Cas9 complex in the reaction solution may be, for example, in the range of 10 to 300 nM. The final concentration of the target DNA in the reaction solution may be in the range of 1 to 1000 nM, for example. The incubation time can be determined as appropriate and may be, for example, 5 minutes to 24 hours.

When the method of this embodiment is carried out in vivo, for example, the inactive CRISPR-Cas9 complex may be introduced into cells containing the target DNA. The cell is not particularly limited as long as it contains the target DNA, and may be a cell of any biological species such as a prokaryote such as Escherichia coli, a fungus such as yeast, an insect, a plant, and an animal. Preferred cells in the method of the present embodiment are cells derived from plants or animals, and particularly preferably cells derived from mammals such as humans. The type of animal cells is also not particularly limited, and cells isolated from any tissue, fertilized eggs, cultured cells and the like can be used. Further, the target DNA contained in the cell may be an endogenous DNA such as genomic DNA or mitochondrial DNA, or an exogenous DNA such as a plasmid vector. The inactive CRISPR-Cas9 complex can be introduced into cells according to a protocol of already established genome editing methods. For example, a pre-prepared guide RNA and an inactivated Cas9 nuclease can be introduced into cells by lipofection, microinjection, electroporation, or the like. Alternatively, an expression vector containing a guide RNA and / or a nucleic acid encoding an inactivated Cas9 nuclease may be introduced into the cell to express the inactive CRISPR-Cas9 complex intracellularly. As the expression vector, an appropriate viral vector or non-viral vector can be selected and used depending on the type of cell into which the inactive CRISPR-Cas9 complex is introduced.

Next, the inactive CRISPR-Cas9 complex is contacted with imidazole or a derivative thereof. As a result, the DNA cleaving activity of Cas9 nuclease is restored, and the CRISPR-Cas9 complex can be put into an activated state.

In the method of the present embodiment, in addition to imidazole, an imidazole derivative having a substituent at the 2-position and / or 4-position of the imidazole ring can be used. When the imidazole ring has substituents at the 2- and 4-positions, both substituents may be the same or different. Examples of the substituent include a substituted or unsubstituted alkyl group, a halogen atom and the like. The alkyl group may be, for example, a C _1-10 alkyl group, preferably a C _1-6 alkyl group, and may be in any form of linear, branched or cyclic. The alkyl group may have one or more hydrogen atoms substituted with a substituent, and examples of the substituent in this case include a halogen atom and the like. The number of substituents and the positions of the substituents in the alkyl group are not particularly limited, but the number of substituents is preferably 0 to 3.

That is, in the method of the present embodiment, imidazole, 4-methylimidazole, 2-methylimidazole, 4-ethylimidazole, 2-ethylimidazole, 2-ethyl-4-methylimidazole, 2-bromoimidazole and the like are preferably used. be able to.

When the method of this embodiment is carried out in vitro, for example, imidazole or a derivative thereof may be added to the above reaction solution and incubated for a certain period of time. When the method of the present embodiment is carried out in vivo, for example, imidazole or a derivative thereof may be added to the cell culture medium and incubated for a certain period of time. The final concentration of imidazole or a derivative thereof in the reaction solution or cell culture solution may be, for example, in the range of 1 to 500 mM, preferably in the range of 10 to 200 mM. The incubation time can be determined as appropriate and may be, for example, 5 minutes to 24 hours.

In the method of this embodiment, it is preferable to then separate imidazole or a derivative thereof from the CRISPR-Cas9 complex. As a result, the DNA-cleaving activity of Cas9 nuclease is lost, and the CRISPR-Cas9 complex can be returned to the inactivated state.

When the method of the present embodiment is carried out in vitro, imidazole or a derivative thereof can be separated from the CRISPR-Cas9 complex by removing imidazole from the reaction solution by, for example, dialysis or ultrafiltration. .. When the method of the present embodiment is carried out in vivo, the imidazole or its derivative is separated from the CRISPR-Cas9 complex, for example, by exchanging with a cell culture medium containing no imidazole or a derivative thereof to remove the imidazole. be able to.

The outline of the method of this embodiment is shown in FIG. The CRISPR-Cas9 complex is homing to the target double-stranded DNA by a guide RNA (“gRNA” in the figure), and in the case of wild-type Cas9, the PAM sequence (“NGG” in the figure) by the HNH and RuvC domains. ), The double-stranded DNA upstream of) is cleaved (Fig. 1A). However, in the case of the Cas9 variant (H840A) in which the 840th histidine residue is replaced with an alanine residue and the imidazole group, which is the side chain of the histidine residue, is deleted, the HNH domain loses its DNA cleavage activity. Since the CRISPR-Cas9 complex can only cleave only one strand of the target double-stranded DNA, genome editing does not occur (FIG. 1B). Here, when imidazole is added from the outside, the DNA cleaving activity of the HNH domain is restored, the CRISPR-Cas9 complex can cleave the target double-stranded DNA, and genome editing becomes possible (FIG. 1C). After that, when imidazole is removed, the CRISPR-Cas9 complex returns to the state shown in FIG. 1B, the DNA cleaving activity of the HNH domain is lost again, and genome editing is stopped. As described above, according to the method of the present embodiment, the DNA cleavage activity of the CRISPR-Cas9 complex can be reversibly controlled, and the genome editing with high accuracy with reduced off-target effect is possible.

According to the second embodiment, the present invention is an inactivated Cas9 nuclease or an inactivated Cas9 nuclease containing a point mutation at the histidine residue corresponding to the 840th position in the numbering based on the amino acid sequence of the Cas9 nuclease derived from pyogenic Cas9 nuclease. A kit for genome editing containing a nucleic acid encoding imidazole or a derivative thereof. The "Cas9 nuclease", "inactivated Cas9 nuclease", and "imidazole or derivative thereof" in this embodiment are the same as those defined in the first embodiment.

In addition to the above configuration, the kit of the present embodiment may further include a buffer and other reagents used for CRISPR-Cas9 genome editing, a manual describing a protocol for CRISPR-Cas9 genome editing, and the like.

The present invention will be further described with reference to examples below. It should be noted that these do not limit the present invention in any way.

<1. Materials and methods>
(1) Preparation of Cas9 variant By the following procedure, the 840th histidine residue of Cas9 nuclease (SEQ ID NO: 12) derived from purulent Lenza bacterium (hereinafter simply referred to as "Cas9") is replaced with an alanine residue. H840A, a variant H840G in which the 840th histidine residue was replaced with a glycine residue, a variant H983A in which the 983th histidine residue was replaced with an alanine residue, and a glycine residue in the 983th histidine residue. A mutant H983G substituted with a group was prepared.

Using the vector pET-Cas9-NLS-His (# 47327, Addgene) containing a nucleic acid encoding a protein with a nuclear localization signal sequence and a histidine tag added to the C-terminal of wild-type Cas9 as a template, the following primer set was used. And then the In-Fusion reaction to obtain expression vectors for Cas9 nickase (Cas9 variant D10A), Cas9 variant H840A, Cas9 variant H840G, Cas9 variant H983A, and Cas9 variant H983G. It was. In the primer sequences shown below, the underlined part indicates the nucleic acid sequence corresponding to the mutant amino acid, and the lower case indicates the nucleic acid sequence modified from the wild-type Cas9 (including silent mutations without modification of the amino acid sequence). ..

(I) Primer set for Cas9 nickase (Cas9 mutant D10A)

(Ii) Primer set for Cas9 mutant H840A

(Iii) Primer set for Cas9 mutant H840G

(Iv) Primer set for Cas9 mutant H983A

(V) Primer set for Cas9 mutant H983G

Escherichia coli BL21 (DE3) was transformed with the obtained expression vector. The obtained transformant was shake-cultured in LB medium at 37 ° C. for 3 hours, IPTG having a final concentration of 0.1 mM was added, and shake-cultured at 18 ° C. overnight. The recovered E. coli was dissolved in lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, 10 mM imidazole, 1 mM benzylsulfonylfluoride, 1 mM dithiothreitol, pH 8.0) and dissolved in Ni-NTA. Purification using a column (30230, QIAGEN) gave a crude product of Cas9 variant. The Cas9 mutant was then purified by gel filtration chromatography and stored at −80 ° C.

(2) Evaluation of DNA-cleaving activity of Cas9 mutant pCX-EGFP (FEBS Lett .; 407 (3): 313-319 (1997), donated by Osaka University) was used as the substrate plasmid DNA. The following sgRNAs targeting the EGFP gene were used as guide RNAs.

sgRNA (EGFP)

Cas9 variant with a final concentration of 30 nM, sgRNA with a final concentration of 30 nM, in a Cas9 reaction buffer (HEPES (pH 8.0) with a final concentration of 20 mM, NaCl with a final concentration of 5 mM, MgCl _{2 with a final concentration of 0.1 mM (pH 8.0)).} And imidazole (pH 8.0) at the concentration shown in the figure was added and incubated at room temperature for 10 minutes. Then, pCX-EGFP having a final concentration of 3 nM was added, and the DNA was cleaved by incubating at 37 ° C. for 1 hour. Then, 6 × Gel Loading Dye, Purple (B7024S, New England Biolabs) was added to the reaction mixture, mixed, and subjected to 0.8% agarose gel electrophoresis. The gel after electrophoresis was stained with GelGreen (41004, Biotium) stain diluted 1/10000 with TAE buffer for 20 minutes, and then photographed with the ChemiDoc XRS + system (Bio-Rad). In addition, as a control, wild-type Cas9 or Cas9 nickase (Cas9 mutant D10A) was used instead of the Cas9 mutant, and the DNA cleavage activity was evaluated by the same procedure.

The following were used for imidazole or its derivative. Imidazole (097-05391, Fujifilm Wako Pure Chemical Industries, Ltd.), 4-Methylimidazole (132-11202, Fujifilm Wako Pure Chemical Industries, Ltd.), 1-methylimidazole (134-21801, Fujifilm Wako Pure Chemical Industries, Ltd.), 2-methylimidazole ( 138-11162, Fujifilm Wako Pure Chemical Industries, Ltd.), 1,2,3-Triazole (320-74161, Fujifilm Wako Pure Chemical Industries, Ltd.), 1,2,4-Triazole (327-91852, Fujifilm Wako Pure Chemical Industries, Ltd.), Pilor (167-05662, Fujifilm Wako Pure Chemical Industries, Ltd.), Pyrazole (165-06903, Fujifilm Wako Pure Chemical Industries, Ltd.).

<2. Restoration of DNA cleavage activity of Cas9 mutant by imidazole>
For the four Cas9 mutants H840A, H840G, H983A and H983G prepared in (1) above, changes in DNA cleavage activity due to the addition of imidazole were evaluated according to the procedure in (2) above. Although the DNA cleavage mechanism of Cas9 has not been elucidated in detail, D839, H840, N854 and N863 in the HNH domain, and D10, E762, H983 and D986 in the RuvC domain are related to the DNA cleavage activity of Cas9. It is presumed to be an amino acid residue.

The results for Cas9 mutants H840A and H840G are shown in FIG. 2, and the results for Cas9 mutants H983A and H983G are shown in FIG. In the figure, "none" is a sample to which Cas9 was not added, that is, the result when the substrate plasmid DNA was not cleaved (in the figure, the band of "plasmid" can be confirmed); "Cas9". Is a sample supplemented with wild-type Cas9, that is, the result when the double strand of the substrate plasmid DNA is cleaved (in the figure, the band of "linear" can be confirmed); "nCas9" is Cas9 nickase. It is a sample to which (Cas9 variant D10A) was added, that is, the result when only one strand of the substrate plasmid DNA was cleaved (in the figure, the band of "nick" can be confirmed).

Neither Cas9 mutants H840A and H840G showed DNA-cleaving activity in the absence of imidazole, but the addition of imidazole restored the DNA-cleaving activity (Fig. 2). On the other hand, the DNA cleavage activity of both Cas9 mutants H983A and H983G did not change even when imidazole was added (FIG. 3). In addition, Cas9 mutant H983G had double-strand breaks in the substrate plasmid DNA even in the absence of imidazole (Fig. 3), confirming that it has DNA cleavage activity. These results suggest that the H840 point mutant may be suitable for the inactivated Cas9 nuclease for controlling DNA cleavage activity by the chemical rescue method.

<3. Examination of imidazole concentration to restore DNA cleavage activity of Cas9 mutant>
For the Cas9 mutants H840A and H840G prepared in (1) above, the DNA cleavage activity in the imidazole concentration range of 0 to 200 mM was evaluated by the procedure in (2) above.

The results are shown in Fig. 4. In both Cas9 mutants H840A and H840G, double-stranded cleavage of the substrate plasmid DNA was observed by the addition of 10 mM imidazole, and the substrate plasmid DNA cleaved in a concentration-dependent manner of imidazole increased. From this result, it was shown that the DNA cleavage activity of the H840 point mutant can be recovered from 10 mM imidazole, and that sufficient DNA cleavage activity can be recovered by imidazole of 50 mM or more.

<4. Recovery of DNA cleavage activity of Cas9 mutant by imidazole over time>
For Cas9 mutants H840A and H840G prepared in (1) above, DNA cleavage was performed according to the procedure in (2) above, except that the imidazole concentration was 100 mM and the incubation time for the DNA cleavage reaction was 0 to 120 minutes. The activity was evaluated.

The results for Cas9 mutant H840A are shown in FIG. 5, and the results for Cas9 mutant H840G are shown in FIG. In both Cas9 mutants H840A and H840G, the amount of substrate plasmid DNA cleaved was increased in an incubation time-dependent manner. In addition, in both Cas9 mutants H840A and H840G, the substrate plasmid DNA cleaved by incubation for 60 minutes or more was significantly increased. Considering that wild-type Cas9 cleaves all substrate DNA in minutes under similar reaction conditions (Science; 361 (6408): 1259-1262 (2018)), imidazole-inactivated Cas9 mutants Chemical rescue was very gradual, which was considered beneficial to facilitate precise control of DNA cleavage activity.

<5. Effect of Magnesium Chloride Concentration on Restoration of DNA Cleaving Activity of Cas9 Mutant by Imidazole>
It has been shown that the HNH domain of Cas9 requires magnesium ions to cleave DNA (Cell; 156 (5): 935-949 (2014)). Therefore, with respect to the Cas9 mutants H840A and H840G prepared in the above (1), the DNA cleavage activity was evaluated by the procedure of the above (2) except _{that the imidazole concentration was 100 mM and the MgCl 2 concentration was 0 to 10 mM.} ..

The results for the Cas9 mutant H840A are shown in FIG. 7, and the results for the Cas9 mutant H840G are shown in FIG. Both Cas9 mutants H840A and H840G showed significant DNA cleavage activity at _{5-10 mM MgCl 2.}

<6. Restoration of DNA cleavage activity of Cas9 mutant by imidazole derivative>
For Cas9 mutants H840A and H840G prepared in (1) above, DNA cleavage activity was evaluated by the procedure in (2) above, except that various imidazole derivatives (100 mM, pH 8.0) were used instead of imidazole. ..

The results for Cas9 mutant H840A are shown in FIG. 9, and the results for Cas9 mutant H840G are shown in FIG. It was confirmed that both Cas9 mutants H840A and H840G can restore DNA cleavage activity by 4-methylimidazole and 2-methylimidazole. From this result, it was shown that the same chemical rescue as imidazole is possible even if an imidazole derivative is used.

Claims (8)

A method for controlling the DNA cleavage activity of Cas9 nuclease, which is a method for controlling the DNA cleavage activity.
(1) The step of contacting the CRISPR-Cas9 ribonuclear protein complex with the target DNA, where the complex is numbered 840th based on the guide RNA and the amino acid sequence of Cas9 nuclease derived from purulent lensaspheres. It contains an inactivated Cas9 nuclease containing a point mutation in the histidine residue corresponding to the position, followed by
(2) A method comprising contacting the complex with imidazole or a derivative thereof. (3) The method according to claim 1, further comprising a step of separating imidazole or a derivative thereof from the complex after the step (2). The method according to claim 1 or 2, wherein the point mutation is a substitution with an alanine residue or a glycine residue. The method according to any one of claims 1 to 3, wherein the imidazole or a derivative thereof has a concentration of 1 to 500 mM. The method according to any one of claims 1 to 4, wherein the imidazole or a derivative thereof is selected from the group consisting of imidazole and an imidazole derivative having a substituent at the 2-position and / or 4-position of the imidazole ring. The method according to any one of claims 1 to 5, which is carried out in vitro. The method according to any one of claims 1 to 5, which is carried out in vivo. Includes inactivated Cas9 nuclease or nucleic acid encoding it, including a point mutation at the histidine residue corresponding to position 840 in the amino acid sequence-based numbering of Cas9 nuclease derived from pyogenic Cas9 nuclease, and imidazole or a derivative thereof. , Kit for genome editing.

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