WO2019103020A1 - 安定で副作用の少ないゲノム編集用複合体及びそれをコードする核酸 - Google Patents
安定で副作用の少ないゲノム編集用複合体及びそれをコードする核酸 Download PDFInfo
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Definitions
- the present invention relates to a stable genome editing complex with few side effects, a nucleic acid encoding the complex, and a genome editing method using the complex.
- Genomic editing is particularly advantageous in prokaryotes, as it does not require integration of the selectable marker gene and can minimize the impact on downstream gene expression in the same operon.
- Phage-derived RecET and ⁇ -Red recombinases have been used as recombinant techniques to facilitate homology-dependent incorporation / substitution of donor DNA or oligonucleotides (eg, Non-Patent Document 1).
- MMR methyl-directed mismatch repair
- highly efficient recombination can be achieved without incorporating a selectable marker (Non-patent Document 2), and multiple target genes within a few days It is used in Multiple Automatic Genome Engineering (MAGE) to generate genetic diversity at the locus.
- MMR methyl-directed mismatch repair
- Non-patent Document 3 Non-patent Document 3
- CRISPR clustered regularly interspaced short palindromic repeats
- Cas CRISPR-related proteins cleave bacterial DNA by cleaving target DNA in a manner dependent on a single guide RNA (sgRNA) and protospacer flanking motif (PAM) It is known to act as an adaptive immune system.
- Cas9 nuclease from Streptococcus pyogenes is widely used as a powerful genome editing tool in eukaryotes with a repair pathway for DNA double strand breaks (DSBs) (e.g. Non-patent Document 4, 5).
- HDR homologous recombination repair
- Non-patent Documents 6 and 7 since current genome editing techniques rely on host DNA repair systems, application to prokaryotes requires further devising. In most bacteria, cell breakage is caused by DNA cleavage by artificial nucleases due to the lack of the NHEJ pathway (Non-patent Documents 6 and 7). Therefore, CRISPR / Cas9 is only used as a counter-selector for cells that have been genetically modified by other methods, such as the ⁇ -Red recombination system (eg, Non-Patent Document 8) , 9).
- deaminase-mediated target base editing which directly edits nucleotides at the target locus without using donor DNA containing homology arms to the target region (e.g. Patent Document 1, Non-patent Document 1) 10-12).
- This technology does not induce bacterial cell death because it utilizes DNA deamination instead of nuclease-mediated DNA cleavage, and is applicable to bacterial genome editing, but its mutation efficiency, particularly multiple The efficiency of simultaneous editing for a part is not sufficient.
- an object of the present invention is to provide a nucleic acid such as a low toxicity vector which can be stably amplified in a host, and a genome editing complex encoded by the nucleic acid, as well as the vector, if necessary. It is applicable to a wide range of bacteria without depending on host dependent factors such as RecA by using a nucleic acid modifying enzyme, and a genome editing method capable of modifying bacterial DNA while suppressing non-specific mutation etc. It is to provide a method.
- the present inventor can stabilize a vector in bacteria by suppressing the abundance of a genome editing complex highly toxic to bacteria as a host in bacteria, and also can cause nonspecific mutation of bacterial DNA, etc.
- I was inspired by the idea of reducing Therefore, in order to suppress the abundance of the genome editing complex, attention is focused on the protein degradation tag LVA tag, which is known to promote the degradation of proteins in bacteria and shorten the half life, and to conduct research.
- LVA tag which is known to promote the degradation of proteins in bacteria and shorten the half life, and to conduct research.
- the proteolysis tag by adding the proteolysis tag to the genome editing complex, it is possible to reduce nonspecific mutations while maintaining the mutation efficiency to the target site, and even when UGI is combined, it is not effective. It was demonstrated that specific mutations can be reduced and target sequences can be modified with high efficiency (FIG. 9, FIG. 10). As a result of further studies based on these findings, the present inventors have completed the present invention.
- a nucleic acid sequence recognition module which specifically binds to a target nucleotide sequence in double-stranded DNA, (i) a peptide containing three hydrophobic amino acid residues at the C-terminus, or (ii) at least one of the amino acid residues
- the complex which the protein degradation tag which consists of a peptide which contains the amino acid 3 residue in which one part was substituted by serine in the C terminus joined.
- the complex is a complex further bound with a nucleic acid modifying enzyme, which converts or deletes one or more nucleotides of the targeted site into one or more other nucleotides, or the targeting The complex according to [1], which inserts one or more nucleotides at the designated site.
- a nucleic acid modifying enzyme which converts or deletes one or more nucleotides of the targeted site into one or more other nucleotides, or the targeting The complex according to [1], which inserts one or more nucleotides at the designated site.
- [3] The complex according to [1] or [2], wherein the amino acid 3 residue is leucine-valine-alanine, leucine-alanine-alanine, alanine-alanine-valine or alanine-serine-valine.
- nucleic acid sequence recognition module is a CRISPR-Cas system in which only one or two of the two DNA cleaving abilities of Cas are inactivated.
- the complex as described in.
- the nucleic acid modifying enzyme is a nucleic acid base converting enzyme or a DNA glycosylase.
- nucleic acid base converting enzyme is a deaminase.
- a method for altering the targeted site of bacterial double-stranded DNA, or modulating the expression of a gene encoded by double-stranded DNA in the vicinity of the site, which is selected double-stranded A nucleic acid sequence recognition module which specifically binds to a target nucleotide sequence in DNA, (i) a peptide containing 3 hydrophobic amino acid residues at the C-terminus, or (ii) at least a portion of the amino acid residues is substituted by serine And D.
- the complex is a complex further bound with a nucleic acid modifying enzyme, which converts or deletes one or more nucleotides of the targeted site into one or more other nucleotides, or the target
- the method according to [10] which comprises the step of inserting one or more nucleotides at the immobilized site.
- [15] The method according to any one of [10] to [14], which comprises using two or more types of nucleic acid sequence recognition modules that specifically bind to different target nucleotide sequences.
- [16] The method according to [15], wherein the different target nucleotide sequences are present in different genes.
- the nucleic acid modifying enzyme is a nucleic acid base converting enzyme or a DNA glycosylase.
- the nucleic acid base converting enzyme is deaminase.
- the contact of double-stranded DNA with a complex is carried out by introducing a nucleic acid encoding the complex into a bacterium having the double-stranded DNA. Method described.
- a low toxic nucleic acid eg, vector
- a genome editing complex encoded by the nucleic acid are provided.
- the method of genome editing using the nucleic acid and nucleic acid modifying enzyme of the present invention it is possible to modify a host bacterial gene while suppressing nonspecific mutation or the like, or to express the gene encoded by double-stranded DNA. It is possible to adjust the This approach is applicable to a wide range of bacteria, as it does not rely on host dependent factors such as RecA.
- FIG. 1 shows a schematic of the Target-AID system in bacteria.
- (a) shows a schematic model of Target-AID (dCas9-PmCDA1 / sgRNA) base editing.
- the dCas9-PmCDA1 / sgRNA complex binds to double stranded DNA to form an R loop in a sgRNA and PAM dependent manner.
- PmCDAl catalyzes the deamination of cytosine located in the upper (non-complementary) strand within the upstream 15-20 bases of PAM, leading to a C to T mutagenesis.
- (b) shows a single Target-AID plasmid of bacteria.
- This plasmid contains the chloramphenicol resistance (Cm R ) gene, temperature sensitive (ts) ⁇ cI repressor, pSC101 origin of replication (ori) and RepA101 (ts).
- the ⁇ operator expresses the dCas9-PmCDA1 fusion at high temperature (> 37 ° C.) as the cI repressor (ts) inactivates.
- sgRNA is expressed by the constitutive promoter J23119.
- dCas9 represents a nuclease-deficient Cas9 having the D10A H840A mutation
- PmCDA1 represents P. marinus (limus) cytosine deaminase.
- FIG. 2 shows the transformation efficiency of Cas9 and Target AID vectors in E. coli.
- E. coli DH5 ⁇ strain is transformed with a plasmid expressing each of the modifying proteins (Cas9, dCas, Cas9-CDA, nCas-CDA or dCas-CDA) together with sgRNA targeting the galK gene, and a chloramphenicol resistance marker was selected.
- Viable cells were counted and calculated as colony forming units (CFU) per amount of transformed plasmid DNA.
- the dots represent three independent experiments, and the boxes represent 95% confidence intervals of the geometric mean by t-test analysis.
- FIG. 3 shows mutations induced at specific sites of the galK9 gene in dCas-CDA.
- DH5 ⁇ cells expressing dCas-CDA with sgRNA targeting galK_9 were spotted on LB agar plates and single colonies were isolated. Eight randomly picked clones were sequenced and the sequences aligned. The translated amino acid sequence is shown at the bottom of each nucleotide sequence. The frequency of each sequence is shown as the number of clones. Box and inverted box indicate target and PAM sequences, respectively. The ORF numbers are shown at the top. The mutated sites are highlighted in black and the mutated bases are highlighted in bold. The mutated codons are underlined.
- FIG. 4 shows mutation frequencies assessed by drug resistance. (a) shows galK mutagenesis and 2-DOG resistance frequency.
- DH5 ⁇ cells expressing dCas-CDA with non-targeted sgRNA (vector) or ssRNA targeting galK_9 were serially diluted, spotted on agar plates of M63 medium with or without 2-DOG and colony counted.
- (b) shows rpoB mutagenesis and rifampicin resistance frequency.
- Cells expressing dCas-CDA with non-targeted sgRNA (vector) or ssRNA targeting rpoB_1 were serially diluted, spotted on LB agar plates with or without rifampicin and colony counted.
- the drug resistance frequency was calculated as the number of drug resistant colonies relative to the number of unselected colonies.
- FIG. 5 shows gain of function mutagenesis of the rpoB gene.
- (a) shows a sequence alignment of rCaB mutations induced by dCas-CDA.
- DH5 ⁇ cells expressing dCas-CDA with sgRNA targeting rpoB_1 were spotted on LB agar plates to isolate single colonies.
- Eight randomly picked clones were sequenced and the sequences aligned.
- the translated amino acid sequence is shown at the bottom of each nucleotide sequence.
- the frequency of each sequence is shown as the number of clones. Boxes and inverted boxes indicate target and PAM sequences.
- Parental / variable mutations include insertions, deletions, single nucleotide variants (SNVs) and multiple nucleotide variants (MNVs), including insertions, deletions, single nucleotide variants (SNVs) and multiple nucleotide variants (MNVs)
- SNVs single nucleotide variants
- MNVs multiple nucleotide variants
- the number of variants obtained by subtracting the mutation of the common parent from the detected variants is shown.
- the mutations detected indicate the number of mutations (count), genomic locus (region / gene), reference genomic sequence (reference) and mutant allele (allele). Variant calling was performed as described in the examples.
- (c) shows the sequence around the detected mutation listed in (b). The mutated sites are highlighted in gray shades and the mutated bases and amino acids are highlighted in bold.
- FIG. 6 shows the presence or absence of UGI-LVA, and the mutation position and frequency when sgRNAs of different lengths were used.
- Test target sequences (galK_8, 9, 11 and 13) over 20 nt in length with dCas-CDA (white bar on left) or dCas-CDA-UGI-LVA (black bar on right) and analyze by deep sequencing did. The average of three independent experiments was plotted. Gray shaded and inverted boxes indicate galK target sequence and PAM, respectively. The mutated bases are underlined.
- FIG. 7 shows the effect of target sequence characteristics on Target-AID induced mutation location and frequency. Cells expressing dCas-CDA and each targeted sgRNA were analyzed by deep sequencing.
- the target sequence (20 nt in length or as indicated) was the (+) or lower (-) DNA strand above the galK ORF, but as expected the missense (M) or nonsense (N) mutation had been introduced .
- the corresponding ORF numbers were indicated (Position).
- the mutation frequency of the base position of the peak was obtained as the average of three independent experiments. Mutation frequencies> 50%, 10-50% or ⁇ 10% were distinguished by gray shades.
- FIG. 8 shows the effect of target length on the mutational spectrum.
- (a) shows the mutation frequency with target sequences of various lengths in gsiA.
- Target sequences containing poly C at the distal site were compiled with dCas-CDA-UGI-LVA and analyzed by deep sequencing.
- Mutation spectra of sgRNAs having a length of 18 nt, 20 nt, 22 nt or 24 nt were distinguished by gray shades. The average of three independent experiments is shown. The inverted box indicates PAM. The mutated bases are underlined.
- (b) shows target mutation frequency in ycbF and yfiH. The target was set to the lower strand.
- the mutation spectrum of sgRNA having a length of 18 nt, 20 nt or 22 nt is shown as in (a).
- (c) shows the averaged mutation spectrum for each sgRNA length of (a) and (b). The peak positions were numbered.
- FIG. 9 shows multiple mutagenesis in the galK gene.
- (a) shows nonspecific mutagenic effects assessed by rifampicin resistance.
- Cells that express each protein (vector, dCas, dCas-CDA, dCas-CDA-LVA or dCas-CDA1-UGI-LVA) with or without rifampicin with tandem-sgRNA units for the galK_10-galK_11-galK13 target
- the cells were spotted on LB agar plates to assess the frequency of nonspecific mutations.
- the dots represent at least three independent experiments, and the boxes represent 95% confidence intervals of the geometric mean by t-test analysis.
- (b) shows the on target multiple mutation frequency induced in the target region.
- the eight randomly selected clones of (a) were sequenced at the three targeted loci to show the frequency of single, double or triple mutant clones.
- (c and d) show sequence alignment of the variants.
- single targets galK_10, galK_11 or galK_13
- c or triple targets were mutated.
- Eight randomly picked clones were sequenced and the sequences aligned. Boxes and inverted boxes indicate target and PAM sequences, respectively. Mutation sites are highlighted in black and bold.
- FIG. 10 shows multiple mutagenesis.
- (a) is a plasmid containing two plasmids for multiplex mutagenesis (a modification vector expressing dCas-CDA-UGI-LVA and two tandem repeat sgRNA-units containing three targeting sgRNAs each)
- the schematic diagram of pSBP80608 is shown.
- (b) shows sequence alignment of the target area. Eight randomly picked clones were sequenced and aligned at each target region. The clone number is indicated to the left of the sequence. Box and inverted box indicate target sequence and PAM. The mutated sites and bases are highlighted in black and bold.
- FIG. 11 shows the simultaneous destruction of multiple copies of the transposase gene.
- FIG. 12 shows a method for isolation and confirmation of IS-edited cells. Clone isolation and sequence confirmation were performed step by step.
- FIG. 13 shows a schematic diagram of the yeast expression vector (background: pRS315 vector) used in Example 5.
- Gal1p indicates GAL1-10 promoter.
- a genome editing complex and a nucleic acid encoding the same provides a genome editing complex in which a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in double-stranded DNA, and a proteolysis tag are bound
- a nucleic acid encoding the complex is provided.
- a complex further bound by a nucleic acid modifying enzyme (ie, a complex wherein a nucleic acid sequence recognition module, a nucleic acid modifying enzyme, and a proteolysis tag are bound)
- a complex capable of altering the nucleic acid at the targeted site is provided.
- the complex in order to improve the efficiency of modification of double-stranded DNA, the complex may further be bound by an inhibitor of base excision repair.
- a transcription regulator may be further bound to the complex.
- a “complex according to the present invention” or a “genome according to the present invention” is collectively including a complex to which at least one of a nucleic acid modifying enzyme, an inhibitor of base excision repair and a transcription regulatory factor is bound. It may be referred to as an editing complex, and a complex to which a nucleic acid modifying enzyme is bound may be particularly referred to as a "nucleic acid modifying enzyme complex”. Also, the nucleic acids encoding these complexes may be collectively referred to as "the nucleic acids of the present invention”.
- nucleic acid of the present invention When the nucleic acid of the present invention is introduced into a host bacterium (eg, E. coli) for the purpose of replication and culture, not for the purpose of modifying the DNA, even when the complex is unintentionally expressed from the nucleic acid, a protein Since the complex is rapidly degraded by the degradation tag, the toxicity to host bacteria can be reduced.
- a host bacterium eg, E. coli
- transformation of the host bacterium is carried out as compared to the case where a nucleic acid encoding a protein degradation tag is not introduced. High efficiency has been demonstrated in the examples below.
- the nucleic acid of the present invention containing a sequence encoding a protein degradation tag can be stably replicated in bacteria as a nucleic acid for genome editing to hosts other than bacteria (eg, eukaryotes). Therefore, it is also useful to add a sequence encoding the protein degradation tag of the present invention to a vector for genome editing in hosts other than bacteria.
- “modification” of double-stranded DNA means that one nucleotide (eg, dC) on the DNA strand is converted or deleted to another nucleotide (eg, dT, dA or dG). Or means that a nucleotide or nucleotide sequence is inserted between certain nucleotides on the DNA strand.
- the double-stranded DNA to be modified is not particularly limited as long as it is double-stranded DNA present in a host cell, but is preferably genomic DNA.
- target site of double-stranded DNA refers to all or part of the “target nucleotide sequence” to which the nucleic acid sequence recognition module specifically recognizes and binds, or to the vicinity of the target nucleotide sequence 5 'upstream and / or 3' downstream).
- target nucleotide sequence means a sequence to which a nucleic acid sequence recognition module in double stranded DNA binds.
- gene editing refers not only to double-stranded DNA modification, but also promotes or suppresses expression of the gene encoded by double-stranded DNA in the vicinity of the targeted site. It is used in the meaning which also includes.
- the "nucleic acid sequence recognition module” means a molecule or molecule complex having the ability to specifically recognize and bind a specific nucleotide sequence on a DNA strand (ie, a target nucleotide sequence).
- a nucleic acid modified enzyme complex binding of the nucleic acid sequence recognition module to the target nucleotide sequence results in targeting of the nucleic acid modified enzyme and / or inhibitor of base excision repair linked to the module to double-stranded DNA. It is possible to act specifically at
- nucleic acid modifying enzyme means an enzyme which modifies a nucleic acid to cause DNA modification directly or indirectly by the modification, and as long as it has catalytic activity, it is a peptide fragment thereof It is also good.
- DNA strand cleavage reaction a reaction that cleaves a DNA strand (hereinafter also referred to as "DNA strand cleavage reaction”) catalyzed by a nucleolytic enzyme, or a cleavage of a DNA strand catalyzed by a nucleic acid base converting enzyme (A reaction which also refers to a “nucleobase conversion reaction”) (for example, a base deamination reaction) which converts a substituent on the purine or pyrimidine ring of a nucleobase into another group or atom, which is a reaction not directly involving A), a reaction catalyzed by DNA glycosylase, a reaction for hydrolyzing N-glycosidic bond of DNA (hereinafter also referred to as “abasic reaction”), and the like.
- a reaction which also refers to a “nucleobase conversion reaction” (for example, a base deamination reaction) which converts a substituent on the purine or
- nucleic acid modified enzyme complex containing a nucleic acid base converting enzyme by adding a proteolysis tag to a nucleic acid modified enzyme complex containing a nucleic acid base converting enzyme, the toxicity of the complex to host bacteria can be reduced. Therefore, the technique of the present invention is applied not only to nucleic acid base converting enzymes but also to genome editing using nucleic acid degrading enzymes, which has conventionally been difficult to apply to bacteria because of its high toxicity. Can. Therefore, as the nucleic acid modifying enzyme used in the present invention, a nucleic acid degrading enzyme, a nucleic acid base converting enzyme, a DNA glycosylase and the like can be mentioned.
- nucleic acid base converting enzymes and DNA glycosylases are preferable, and by using these enzymes, at least one strand of double-stranded DNA is not cut at the targeted site, The targeted site can be modified.
- the “proteolytic tag” is mainly composed of a peptide containing three or more hydrophobic amino acid residues, and by adding to the genome editing complex, the protein is halved as compared to the non-added protein. It means a peptide whose period is short.
- amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, and the protein degradation tag of the present invention even contains any of these three amino acid residues at the C-terminus
- the other constitution and it may be a peptide consisting of three amino acid residues.
- a peptide in which some or all of the hydrophobic amino acid residues are substituted with serine or threonine is also included in the protein degradation tag of the present invention.
- the preferred three amino acid residues are not particularly limited, but high efficacy was observed in E. coli (E. coli) and Pseudomonas putida (Andersen JB et al., Apll. Environ. Microbiol., 64: 2240-2246).
- a proteolysis tag containing these three amino acid residues is a database of tm RNA tag peptides (eg, tmRDB, http://www.ag.auburn.edu/mirror/tmRDB/peptide/peptidephylolist.html), etc. You can refer to it.
- YAASV SEQ ID NO: 324), YALAA (SEQ ID NO: 325), ANDENYALAA (SEQ ID NO: 181) and AANDENYALAA (SEQ ID NO: 182) known as tmRNA tag peptides of E. coli are known as tmRNA tag peptides of Bacillus spp.
- GKQNNLSLAA SEQ ID NO: 183
- GKSNNNFALAA SEQ ID NO: 184
- GKENNNFALAA SEQ ID NO: 185
- GKTNSFNQNVALAA SEQ ID NO: 186
- GKSNQNLALAA SEQ ID NO: 187
- GKQNYALAA SEQ ID NO: 188
- ANDDQYGAALAA (SEQ ID NO: 190), ANDENYGQEFALAA (SEQ ID NO: 191), ANDETY GDYALAA (SEQ ID NO: 192), ANDETYGEYALAA (SEQ ID NO: 193), ANDETYGEETYALAA (SEQ ID NO: 194), ANDENYGAEYKLAA (SEQ ID NO: 195) And ANDENYGAQLAA (SEQ ID NO: 196), but not Streptococcus sp.
- AKNTNSYALAA SEQ ID NO: 197
- AKNTNSYAVAA SEQ ID NO: 198
- AKNNTTYALAA SEQ ID NO: 199
- AKNTNTYALAA SEQ ID NO: 200
- AKNNTSYALAA SEQ ID NO: 201
- Proteolytic tags typically consist of 3-15 amino acid residues, but are not limited to this range. In one embodiment, the proteolytic tag consists of 3 to 5 amino acid residues.
- Those skilled in the art can appropriately select a proteolysis tag according to the type of host bacteria and the like. In the present specification, unless otherwise specified, upper case letters of the alphabet indicate single-letter designations for amino acids, and amino acid sequences are written from left to right in the N-terminal to C-terminal direction.
- a genome editing complex means a nucleic acid modifying activity or expression to which a specific nucleotide sequence recognition ability is imparted, comprising a complex in which the above-mentioned nucleic acid sequence recognition module and a proteolysis tag are linked.
- nucleic acid modified enzyme complex means a molecular complex having regulatory activity
- nucleic acid modified enzyme complex includes a complex in which the nucleic acid sequence recognition module, the nucleic acid modifying enzyme and the proteolysis tag are linked. It means a molecular complex having a nucleic acid modifying activity to which a nucleotide sequence recognition ability is imparted.
- the complex may further be linked to an inhibitor of base excision repair.
- complex includes not only those composed of a plurality of molecules but also those having a molecule constituting the above-mentioned complex of the present invention in a single molecule, such as a fusion protein .
- a proteolysis tag is bound to a molecule or molecular complex in which a nucleic acid sequence recognition module and a nucleic acid modifying enzyme function together are also included in the complex of the present invention Is included.
- encoding a complex includes both encoding each of the molecules that make up the complex and encoding a fusion protein that has the molecules that make up the molecule in a single molecule.
- the nucleic acid degrading enzyme used in the present invention is not particularly limited as long as it can catalyze the above reaction, and, for example, nuclease (eg, Cas effector protein (eg, Cas9, Cpf1), endonuclease (eg, restriction enzyme) , Exonuclease etc.), recombinase, DNA gyrase, DNA topoisomerase, transposase etc.
- nuclease eg, Cas effector protein (eg, Cas9, Cpf1)
- endonuclease eg, restriction enzyme
- recombinase eg, DNA gyrase, DNA topoisomerase, transposase etc.
- the nucleic acid base converting enzyme used in the present invention is not particularly limited as long as it can catalyze the above reaction, and for example, a nucleic acid / nucleotide deaminase super that catalyzes a deamination reaction for converting an amino group to a carbonyl group.
- a nucleic acid / nucleotide deaminase super that catalyzes a deamination reaction for converting an amino group to a carbonyl group.
- deaminases belonging to the family.
- cytidine deaminase capable of converting cytosine or 5-methylcytosine to uracil or thymine
- adenosine deaminase capable of converting adenine to hypoxanthine
- guanosine deaminase capable of converting guanine to xanthine
- AID activation-induced cytidine deaminase which is an enzyme for introducing a mutation into an immunoglobulin gene in acquired immunity of a vertebrate is mentioned.
- the origin of the nucleobase converting enzyme is not particularly limited.
- PmCDA1 Petromyzon marinus cytosine deaminase 1
- AID Activation-induced cytidine
- AICDA Activation-induced cytidine
- the nucleotide sequence and amino acid sequence of PmCDA1 cDNA can be referred to GenBank accession Nos. EF094822 and ABO15149
- the nucleotide sequence and amino acid sequence of human AID cDNA can be referred to GenBank accession Nos. NM — 02661 and NP — 065712, respectively.
- PmCDA1 is preferred from the viewpoint of the enzyme activity.
- the DNA glycosylase used in the present invention is not particularly limited as long as it can catalyze the above reaction, and thymine DNA glycosylase, oxoguanine glucosylase, alkyl adenine DNA glycosylase (eg, yeast 3-methyladenine-DNA glycosylase) And the like.
- the present inventors previously reduced cytotoxicity by using DNA glycosylase, which has sufficiently low reactivity with unrelaxed double-stranded DNA (unrelaxed DNA), to reduce the cytotoxicity and efficiently target sequences. It has been reported that it can be modified (WO 2016/072399).
- the DNA glycosylase a DNA glycosylase having a sufficiently low reactivity to the unstrained double helix DNA.
- a DNA glycosylase a variant of UNG (uracil-DNA glycosylase) having cytosine-DNA glycosylase (CDG) activity and / or thymine-DNA glycosylase (TDG) activity described in WO 2016/072399, Examples include UDG mutants derived from vaccinia virus.
- mutant of UNG include N222D / L304A double mutant, N222D / R308E double mutant, N222D / R308C double mutant, Y164A / L304A double mutant, Y164A / R308E double mutant of yeast UNG1.
- a variant in which a similar mutation is introduced to the amino acids corresponding to each of the above variants may be used.
- vaccinia virus-derived UDG mutants N120D mutant, Y70G mutant, Y70A mutant, N120D / Y70G double mutant, N120D / Y70A double mutant and the like can be mentioned.
- a DNA glycosylase divided into two fragments, wherein each fragment is bound to one of two divided nucleic acid sequence recognition modules to form two complexes, and both complexes
- a split enzyme designed to allow the nucleic acid sequence recognition module to specifically bind to a target nucleotide sequence upon folding, which allows the DNA glycosylase to catalyze an abasic reaction. It may be.
- Split enzymes are designed and produced in accordance with, for example, the description of WO 2016/072399, Nat Biotechnol. 33 (2): 139-142 (2015), PNAS 112 (10): 298 4-2989 (2015). can do.
- base excision repair is one of the DNA repair mechanisms possessed by an organism, and means a mechanism that repairs base damage by excising and rejoining a base-damaged part with an enzyme. Removal of damaged bases is carried out by DNA glycosylase which is an enzyme that hydrolyzes N-glycosidic bond of DNA, and a base-free site resulting from the abasic reaction by the enzyme (apurinic / apyrimidic (AP) site) Is processed by enzymes downstream of the base excision repair (BER) pathway, such as AP endonuclease, DNA polymerase, DNA ligase.
- DNA glycosylase is an enzyme that hydrolyzes N-glycosidic bond of DNA, and a base-free site resulting from the abasic reaction by the enzyme (apurinic / apyrimidic (AP) site) Is processed by enzymes downstream of the base excision repair (BER) pathway, such as AP endonuclease, DNA polymerase, DNA
- UNG NM_003362
- SMUG1 NM_014311
- MBD4 NM_003925
- TDG NM_003211
- OGG1 NM_002542
- MYH NM_012222
- NTHL1 NM_002528
- MPG NM_002434
- NEIL1 NM_024608
- NEIL2 NM_140543
- NEIL3 NM_018248
- APE1 NM_001641
- APE2 NM_014481
- LIG3 NM_013975
- XRCC1 NM_006297
- ADPRT PARP1
- ADPRT2 NM_0016718
- ADPRT2 NM_00544
- an inhibitor of base excision repair inhibits either of the above-mentioned BER pathways or inhibits BER as a result by inhibiting the expression itself of molecules recruited to the BER pathway.
- Mean proteins that The inhibitor of base excision repair used in the present invention is not particularly limited as long as it inhibits BER as a result, but from the viewpoint of efficiency, inhibitors of DNA glycosylase located upstream of the BER pathway are preferred.
- the inhibitors of DNA glycosylase used in the present invention include inhibitors of thymine DNA glycosylase, inhibitors of uracil DNA glycosylase, inhibitors of oxoguanine DNA glycosylase, inhibitors of alkylguanine DNA glycosylase, etc. It is not restricted.
- cytidine deaminase when using cytidine deaminase as a nucleic acid modifying enzyme, it is suitable to use an inhibitor of uracil DNA glycosylase in order to inhibit repair of U: G or G: U mismatch of DNA generated by mutation.
- a uracil DNA glycosylase inhibitor (UGI) derived from PBS1 which is a Bacillus subtilis bacteriophage
- a uracil DNA glycosylase inhibitor (UGI) derived from PBS2 which is a Bacillus subtilis bacteriophage (Wang, Z., and Mosbaugh, D. W. (1988) J. Bacteriol. 170, 1082-1091), but not limited thereto, as long as the above-mentioned DNA mismatch repair inhibitor is used, It can be used in the present invention.
- UGI derived from PBS2 is also known to have effects of mutation and cleavage other than C to T on DNA and resistance to recombination, so it is suitable to use UGI derived from PBS2.
- the AP endonuclease nicks the abasic site (AP site), and the exonuclease completely removes the AP site Be done.
- DNA polymerase creates a new base using the base of the opposite strand as a template, and finally the DNA ligase fills in the nick to complete the repair.
- Mutant AP endonucleases that have lost enzyme activity but retain the ability to bind to the AP site are known to competitively inhibit BER. Thus, these mutant AP endonucleases can also be used as inhibitors of the base excision repair of the present invention.
- mutant AP endonuclease is not particularly limited, for example, AP endonuclease derived from E. coli, yeast, mammals (eg, human, mouse, pig, cow, horse, monkey, etc.) can be used.
- the amino acid sequence of human Ape1 can be referred to as Uniprot KB No. P27695.
- mutant AP endonucleases that have lost enzyme activity but retain the ability to bind to the AP site include proteins in which the active site or the cofactor Mg binding site has been mutated.
- E96Q, Y171A, Y171F, Y171H, D210N, D210A, N212A and the like can be mentioned.
- transcription regulatory factor means a protein or a domain having an activity promoting or suppressing transcription of a target gene, and hereinafter, “transcription activation” is carried out having an activity promoting transcription.
- a factor “and one having an activity to suppress transcription may be referred to as” transcriptional repressor ".
- the transcriptional activation factor used in the present invention is not particularly limited as long as it can promote transcription of a target gene, and examples thereof include the activation domain of HSV (Herpes simplex virus) VP16, p65 subunit of NF ⁇ B. , VP64, VP160, HSF, P300 and EB virus (Epstein-Barr Virus) RTA, and their fusion proteins.
- the transcriptional repressor used in the present invention is not particularly limited as long as it can repress transcription of a target gene, and includes, for example, KRAB, MBD2B, v-ErbA, SID (concatemer of SID (SID4X) And MBD2, MBD3, DNMT family (eg, DNMT1, DNMT3A, DNMT3B), Rb, MeCP2, ROM2 and AtHD2A, and their fusion proteins.
- the target nucleotide sequence in double-stranded DNA recognized by the nucleic acid sequence recognition module of the complex of the present invention is not particularly limited as long as the module can specifically bind, and any sequence in double-stranded DNA It may be.
- the length of the target nucleotide sequence need only be sufficient for the nucleic acid sequence recognition module to specifically bind, eg depending on the size of the genome when introducing mutations at specific sites in mammalian genomic DNA It is 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 17 nucleotides or more.
- the upper limit of the length is not particularly limited, but is preferably 25 nucleotides or less.
- a nucleic acid sequence recognition module of the complex of the present invention for example, a CRISPR-Cas system (hereinafter also referred to as “Crisps-mutated Cas”) in which at least one DNA cleaving ability of Cas effector protein is inactivated, zinc finger motif, TAL
- Crisps-mutated Cas in which at least one DNA cleaving ability of Cas effector protein is inactivated, zinc finger motif, TAL
- restriction enzymes, transcriptional regulatory factors, fragments containing a DNA binding domain of a protein capable of specifically binding to DNA such as RNA polymerase, etc. may be used, but not limited thereto.
- CRISPR-Cas system in which a nucleic acid sequence recognition module and a nucleic acid modifying enzyme are integrated (Cas effector protein of the system maintains both activities of DNA cleavability).
- Cas effector protein of the system maintains both activities of DNA cleavability.
- CRISPR-mutated Cas zinc finger motif, TAL effector, PPR motif and the like can be mentioned.
- the zinc finger motif is a linkage of 3 to 6 different zinc finger units of Cys2His2 type (one finger recognizes about 3 bases), and can recognize a target nucleotide sequence of 9 to 18 bases.
- Zinc finger motifs can be obtained by the modular assembly method (Nat Biotechnol (2002) 20: 135-141), the OPEN method (Mol Cell (2008) 31: 294-301), the CoDA method (Nat Methods (2011) 8: 67-69). And E. coli one-hybrid method (Nat Biotechnol (2008) 26: 695-701).
- Patent No. 4968498 for details of the production of zinc finger motifs.
- the TAL effector has a repeating structure of modules of about 34 amino acids, and the binding stability and base specificity are determined by the 12th and 13th amino acid residues of one module (referred to as RVD). Ru. Since each module is highly independent, it is possible to create a TAL effector specific to a target nucleotide sequence simply by joining the modules. TAL effectors are prepared using open resources (REAL method (Curr Protoc Mol Biol (2012) Chapter 12: Unit 12.15), FLASH method (Nat Biotechnol (2012) 30: 460-465), Golden Gate method (Nucleic Acids) Res (2011) 39: e82) etc.) have been established, and it is possible to design a TAL effector to a target nucleotide sequence relatively easily. For details of production of TAL effector, reference can be made to JP-A-2013-513389.
- the PPR motif is constructed to recognize a specific nucleotide sequence by a series of PPR motifs consisting of 35 amino acids and recognizing one nucleobase, and the 1st, 4th and ii (-2) th amino acids of each motif Recognize the target base only. Since there is no dependence on the motif configuration and there is no interference from the motifs on both sides, it is possible to produce a PPR protein specific for a target nucleotide sequence simply by joining PPR motifs like TAL effector. Japanese Patent Application Laid-Open No. 2013-128413 can be referred to for details of preparation of the PPR motif.
- DNA binding domain of these proteins is well known and, for example, a fragment containing the domain and having no ability to cleave DNA double strand It can be easily designed and built.
- any of the above nucleic acid sequence recognition modules can also be provided as a fusion protein with the above nucleic acid modifying enzyme and / or an inhibitor of base excision repair, or SH3 domain, PDZ domain
- a protein binding domain such as GK domain or GB domain and its binding partner are respectively fused to a nucleic acid sequence recognition module and a nucleic acid modifying enzyme and / or an inhibitor for base excision repair, and the domain and its binding partner It may be provided as a protein complex through interaction.
- inteins may be fused respectively to the nucleic acid sequence recognition module and the nucleic acid modifying enzyme and / or the inhibitor of base excision repair, and the two may be linked by ligation after each protein synthesis.
- the protein degradation tag may be bound to any of the components of the nucleic acid modified enzyme complex (nucleic acid sequence recognition module, nucleic acid modified enzyme and inhibitor of base excision repair), and may be bound to a plurality of component molecules.
- the transcriptional regulatory factor may be provided as a fusion protein with a nucleic acid sequence recognition module, or a nucleic acid via the protein binding domain as described above and their binding partners. It may be coupled to a recognition module.
- the proteolysis tag may be bound as a fusion protein, as described above, or may be linked to the genome editing complex or a component thereof via the protein binding domain described above and their binding partners.
- the protein degradation tag is preferably bound to the C-terminus of the genome editing complex or its constituent molecule.
- the nucleic acid of the present invention comprises a nucleic acid sequence recognition module, a protein degradation tag, and optionally, a nucleic acid modifying enzyme and / or an inhibitor for base excision repair, or a transcriptional regulatory factor, as a nucleic acid encoding their fusion protein, Alternatively, they can be prepared as nucleic acids encoding them in such a form that they can form complexes in a host cell after translation into proteins using binding domains, inteins and the like.
- the nucleic acid may be DNA or RNA.
- DNA preferably double-stranded DNA is provided in the form of an expression vector placed under the control of a functional promoter in the host cell.
- RNA it is preferably single stranded RNA.
- DNA encoding nucleic acid sequence recognition modules such as zinc finger motifs, TAL effectors and PPR motifs can be obtained by any of the methods described above for each module.
- a DNA encoding a sequence recognition module such as a restriction enzyme, a transcriptional regulatory factor, an RNA polymerase, etc. is, for example, based on their cDNA sequence information, a region encoding a desired portion (portion containing a DNA binding domain) of the protein Oligo DNA primers can be synthesized so as to cover, and cloning can be performed by amplification by RT-PCR using total RNA or mRNA fractions prepared from cells producing the protein as a template.
- a DNA encoding a nucleic acid-modifying enzyme and an inhibitor of base excision repair is synthesized from an oligo DNA primer based on the cDNA sequence information of the enzyme used, and a total RNA or mRNA fraction prepared from cells producing the enzyme. It is possible to clone by amplifying by RT-PCR method using min as a template.
- DNA encoding UGI derived from PBS2 is designed by designing appropriate primers for the upstream and downstream of CDS based on the DNA sequence (accession No. J04434) registered in the NCBI / GenBank database. It can be cloned from the derived mRNA by RT-PCR.
- the cloned DNA may be digested as it is or optionally with a restriction enzyme, or an appropriate linker (eg, GS linker, GGGAR linker, etc.), spacer (eg, FLAG sequence, etc.) and / or nuclear localization signal (NLS) (If the double-stranded DNA of interest is mitochondrial or chloroplast DNA, each organelle transfer signal) can be added to prepare a DNA encoding a protein. Furthermore, it can be further ligated with DNA encoding a nucleic acid sequence recognition module to prepare DNA encoding a fusion protein.
- a restriction enzyme eg, GS linker, GGGAR linker, etc.
- spacer eg, FLAG sequence, etc.
- NLS nuclear localization signal
- the DNA encoding the genome editing complex of the present invention is chemically synthesized, or the synthesized partially overlapping oligo DNA short strands are connected using PCR or Gibson Assembly. It is also possible to construct a DNA encoding the full length.
- the advantage of constructing full-length DNA in combination with chemical synthesis or the PCR method or Gibson Assembly method is that the codons used can be designed over the entire CDS length according to the host into which the DNA is to be introduced. By converting the DNA sequence into a codon frequently used in the host organism upon expression of heterologous DNA, an increase in the amount of protein expression can be expected.
- codon usage frequency in the host used is, for example, the genetic code usage frequency database (http://www.kazusa.or.jp/codon/index.html) published on the homepage of Kazusa DNA Research Institute (http://www.kazusa.or.jp/codon/index.html). ) May be used, or reference may be made to the literature describing codon usage in each host. Referring to the obtained data and the DNA sequence to be introduced, converting among the codons used for the DNA sequence that are less frequently used in the host into the more frequently used codons encoding the same amino acid Good.
- An expression vector containing DNA encoding the complex of the present invention can be produced, for example, by ligating the DNA downstream of a promoter in a suitable expression vector.
- Expression vectors include E. coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13); Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194); yeast-derived plasmids (eg, pSH19, pSH15); insect cell expression Plasmids (eg: pFast-Bac); animal cell expression plasmids (eg: pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophages such as ⁇ phage; insect virus vectors such as baculovirus ( Examples: BmNPV, AcNPV); Animal virus vectors such as retrovirus, vac
- the promoter may be any promoter as long as it is appropriate for the host used for gene expression.
- a nucleic acid degrading enzyme as a nucleic acid modifying enzyme
- a nucleic acid base converting enzyme and a DNA glycosylase as a nucleic acid modifying enzyme, or without using a nucleic acid modifying enzyme, sufficient cell growth can be obtained even if the complex of the present invention is expressed. Can also be used without restriction.
- SR ⁇ promoter when the host is an animal cell, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex Viral thymidine kinase) promoter or the like is used. Among them, CMV promoter, SR ⁇ promoter and the like are preferable.
- E E.
- coli a J23 series promoter (eg, J23119 promoter), trp promoter, lac promoter, lac promoter, recA promoter, ⁇ P L promoter, lpp promoter, T7 promoter and the like are preferable.
- the SPO1 promoter, the SPO2 promoter, the penP promoter and the like are preferable.
- the host is a yeast, Gal1 / 10 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable.
- polyhedrin promoter, P10 promoter and the like are preferable.
- CaMV 35 S promoter, CaMV 19 S promoter, NOS promoter and the like are preferable.
- expression vectors may optionally contain enhancers, splicing signals, terminators, poly A addition signals, drug resistance genes, selection markers such as auxotrophic complementation genes, replication origins, etc. Can.
- RNA encoding the complex of the present invention can be prepared, for example, by transcribing into mRNA in an in vitro transcription system known per se, using a vector containing DNA encoding each protein as a template.
- the host bacterium used to replicate the nucleic acid of the present invention is not particularly limited as long as it is a bacterium having a protein degradation system using tmRNA (ssrA), and examples thereof include Escherichia bacteria, Bacillus bacteria, and Pseudomonas bacteria ( Examples: Pseudomonas putida), Streptococcus (eg Streptococcus), Streptomyces sp., Vibrio spp., Yersinia sp., Acinetobacter sp., Klebsiella sp., Bordetella sp., Lactococcus sp.
- Escherichia bacteria include Escherichia coli K12 ⁇ DH1 [Proc. Natl. Acad. Sci.
- nucleic acid base converting enzyme or DNA glycosylase is used as the nucleic acid modifying enzyme
- the nucleic acid modifying enzyme and / or the inhibitor of base excision repair is provided as a complex with the mutant Cas by the same method as the linkage with zinc finger etc. Be done.
- the nucleic acid base converting enzyme and / or the inhibitor of base excision repair and the mutant Cas can be bound to the RNA aptamer MS2F6, PP7, etc. using RNA scaffolds by their binding proteins.
- the DNA cleavage site recognition sequence PAM proto-spacer ajacent motif
- PAM is NGG (N is A nucleobase converting enzyme that recognizes any base) 3 bases and can theoretically be targeted anywhere on the genome but can not cleave one or both DNAs and is linked to a mutant Cas
- N is A nucleobase converting enzyme that recognizes any base
- nucleobase conversion or abasic acid occurs at the targeted site (can be appropriately adjusted within the range of several hundred bases including all or a part of the target nucleotide sequence), and a mismatch in double stranded DNA
- cytidine deaminase such as PmCDAl or AID is used as a nucleobase converting enzyme, it may be on the sense or antisense strand of the targeted site Cytosine is converted into
- Errors in the BER system of cells attempting to repair this introduce various mutations.
- the base of the opposite strand is repaired so as to pair with the base of the converted strand without correct repair of the mismatch or abasic (TA or AT in the above example), and so on.
- TA or AT mismatch or abasic
- various mutations are introduced.
- inhibitors of base excision repair intracellular BER mechanisms are inhibited, the frequency of repair errors can be increased, and the efficiency of mutagenesis can be improved.
- the zinc finger motif does not have a high efficiency of producing zinc fingers that specifically bind to a target nucleotide sequence, and the selection of zinc fingers with high binding specificity is complicated, producing a large number of zinc finger motifs that actually function. It is not easy.
- TAL effectors and PPR motifs have higher freedom in target nucleic acid sequence recognition than zinc finger motifs, they need to be designed and constructed each time a large protein depending on the target nucleotide sequence, so there is a problem in efficiency. Will remain.
- the CRISPR-Cas system recognizes a double-stranded DNA sequence of interest by a guide RNA complementary to the target nucleotide sequence, thereby synthesizing an oligo DNA that can specifically hybridize to the target nucleotide sequence. By doing so, any sequence can be targeted. Therefore, in a more preferred embodiment of the present invention, as a nucleic acid sequence recognition module, the CRISPR-Cas system in which both activities of DNA cleaving ability are maintained, or the inactivation of only one or both of the DNA cleaving ability is inactivated The CRISPR-Cas system (Crisps-mutant Cas) is used.
- the nucleic acid sequence recognition module of the present invention using CRISPR-mutated Cas is trans-activating which is necessary for recruitment of CRISPR-RNA (crRNA) containing a sequence complementary to the target nucleotide sequence, and, if necessary, mutant Cas effector protein RNA (tracrRNA) and (if tracrRNA is required, it can be provided as a chimeric RNA with crRNA), provided as a complex with a mutant Cas effector protein.
- An RNA molecule consisting of a crRNA alone or a chimeric RNA of crRNA and tracrRNA, which constitutes a nucleic acid sequence recognition module in combination with a mutant Cas effector protein, is collectively referred to as "guide RNA". The same applies to the case of using the CRISPR / Cas system which has not introduced a mutation.
- the Cas effector protein used in the present invention is not particularly limited as long as it can form a complex with the guide RNA to recognize and bind the target nucleotide sequence in the target gene to the protospacer adjacent motif (PAM) adjacent thereto.
- PAM protospacer adjacent motif
- Is preferably Cas9 or Cpf1.
- Cas9 examples include Cas9 derived from Streptococcus pyogenes (SpCas9; PAM sequence NGG (N is A, G, T or C, the same shall apply hereinafter)), Cas9 derived from Streptococcus thermophilus (StCas9; PAM sequence NNAGAAW), Cas9 (NmCas9; PAM sequence NNNNGATT) derived from Neisseria meningitidis, and the like, but not limited thereto.
- SpCas9 Streptococcus pyogenes
- PAM sequence NGG N is A, G, T or C, the same shall apply hereinafter
- Cas9 derived from Streptococcus thermophilus StCas9; PAM sequence NNAGAAW
- Cas9 NmCas9; PAM sequence NNNNGATT
- it is SpCas9 less restricted by PAM (substantially 2 bases, and theoretically can be targeted almost anywhere on the
- Cpf1 for example, Cpf1 (FnCpf1; PAM sequence NTT) derived from Francisella novisida (Francisella novicida), Cpf1 (AsCpf1; PAM sequence NTTT) derived from Acidaminococcus sp. Examples thereof include Cpf1 (LbCpf1; PAM sequence NTTT) derived from (Lachnospiraceae bacteria) and the like, but are not limited thereto.
- mutant Cas effector protein (sometimes abbreviated as a mutant Cas) used in the present invention, one in which the cleaving ability of both strands of the double-stranded DNA of Cas effector protein is inactivated, and the cleaving ability of one strand are Although it only has inactivated nickase activity, any can be used.
- the 10th Asp residue is converted to an Ala residue, which lacks the ability to cleave the opposite strand of the strand forming the complementary strand with the guide RNA (thus, for the strand forming the complementary strand with the guide RNA D10A mutant (having nickase activity) or the ability to cleave a strand that forms a complementary strand with the guide RNA, in which the His residue at position 840 is converted to an Ala residue (thus, forms a complementary strand with the guide RNA) It is possible to use the H840A mutant (having nickase activity on the opposite strand of the strand), as well as its double variant (dCas9).
- Other mutant Cass can be used as long as they are incapable of cleaving at least one strand of double-stranded DNA.
- mutant Cas is an amino acid residue at a site important for DNA cleavage activity (for example, 10th in the case of SpCas9), using a site-directed mutagenesis method known per se to DNA encoding cloned Cas. Mutation such as, but not limited to), for example, but not limited to, for example, but not limited to, the Asp residue and His residue at position 840, and in the case of FnCpf1, such as the 917th Asp residue and the 1006 position Glu residue.
- a DNA encoding a Cas effector protein may be used in combination with a chemical synthesis or a PCR method or a Gibson Assembly method by a method similar to that described above for a DNA encoding a nucleic acid sequence recognition module and a DNA encoding a DNA glycosylase It can also be constructed as a DNA with codon usage suitable for expression in host cells.
- the obtained Cas effector protein, nucleic acid modifying enzyme, inhibitor of base excision repair, and / or DNA encoding a transcriptional regulatory factor may be inserted downstream of the same expression vector promoter as that described above. it can.
- the DNA encoding the guide RNA is a crRNA sequence (for example, as a Cas effector protein) including a nucleotide sequence complementary to the target nucleotide sequence (also referred to herein as “targeting sequence”).
- a crRNA containing SEQ ID NO: 19; AAUU UCUAC UGUU GUAGA U on the 5 'side of the targeting sequence can be used, and the underlined sequences form a base pair to form a stem-loop structure) Coding sequence or crRNA coding sequence and, if necessary, known tracrRNA coding sequence (for example, tratRNA coding sequence when recruiting Cas9 as Cas effector protein, gttttagagctagaaatagcaagttaaaataaggctagtccactatggaaaagtggcaccgagtcgggtgcttttt; SEQ ID NO: 18) Design and chemically using a DNA / RNA synthesizer It can be synthesized.
- tracrRNA coding sequence for example, tratRNA coding sequence when recruiting Cas9 as Cas effector protein, gttttagagctagaaatagcaagttaaaataaggctagtcca
- target strand means the strand that hybridizes to the crRNA of the target nucleotide sequence, and the opposite strand is a strand that becomes single stranded by hybridization between the target strand and crRNA. It is called (non-targeted strand).
- the target nucleotide sequence is represented by one strand (for example, when representing a PAM sequence) And when representing the positional relationship between the target nucleotide sequence and PAM etc.), it is assumed that the sequence of the non-target strand is represented.
- the length of the targeting sequence is not particularly limited as long as it can specifically bind to the target nucleotide sequence, and is, for example, 15 to 30 nucleotides, preferably 18 to 25 nucleotides.
- the choice of target nucleotide sequence is limited by the presence of PAM flanking the 3 '(in the case of Cas9) or 5' (in the case of Cpf1) of said sequence but according to the findings demonstrated in the examples below.
- the target nucleotide sequence has a regularity such that C tends to be displaced toward the 5 'end as the target nucleotide sequence becomes longer.
- the site of the base capable of introducing a mutation can be shifted. This makes it possible to at least partially relieve the restriction by PAM (NGG in SpCas9), which further increases the freedom of mutagenesis.
- targeting sequence design uses a public guide RNA design website (Crisp Design Tool, CRISPR direct, etc.) to select PAM (for example, SpCas9) from among the CDS sequences of the target gene.
- PAM for example, SpCas9
- NGG the 20mer sequence adjacent to the 3 'side is listed up, and when C within 7 nucleotides in the 3' direction from the 5 'end is converted to T, an amino acid change is made to the protein encoded by the target gene. It can be done by selecting the sequence as it occurs.
- sequence can be selected suitably.
- candidate sequences with a small number of off-target sites in the target host genome can be used as targeting sequences. If the guide RNA design software used does not have the function of searching for off-target sites of the host genome, for example, the host genome for 8 to 12 nucleotides 3 'of the candidate sequence (seed sequence having high ability to discriminate target nucleotide sequence). You can search for off-target sites by applying Blast search against.
- a DNA encoding a guide RNA can also be inserted into the same expression vector as described above, but as a promoter, a pol III promoter (eg, SNR6, SNR52, SCR1, RPR1, U3, U6, H1 promoter, etc.) And terminators (eg, poly T sequences (T 6 sequences etc.)) are preferably used.
- a promoter eg, SNR6, SNR52, SCR1, RPR1, U3, U6, H1 promoter, etc.
- terminators eg, poly T sequences (T 6 sequences etc.) are preferably used.
- the DNA encoding the guide RNA is a sequence complementary to the target strand of the target nucleotide sequence and a known tracrRNA sequence (when recruiting Cas9) or a direct repeat sequence of the crRNA (Cpf1) (In the case of recruiting) can be designed and chemically synthesized using a DNA / RNA synthesizer.
- the complex or the nucleic acid of the present invention described in the present invention is introduced into a host, particularly a bacterium, and the target site of double-stranded DNA in the host is altered or targeted by culturing the host.
- the expression of the gene encoded by double stranded DNA can be regulated in the vicinity of
- a nucleic acid modified enzyme complex is contacted with double stranded DNA of a host bacterium to convert or delete one or more nucleotides of the targeted site to one or more other nucleotides.
- a method of modifying a targeted site of bacterial double-stranded DNA which comprises the step of inserting one or more nucleotides into the targeted site. Be done.
- a nucleic acid base converting enzyme or a DNA glycosylase as a nucleic acid modifying enzyme, it is possible to modify a targeted site without breaking at least one strand of double-stranded DNA at the targeted site.
- a method of contacting a complex of the invention with double stranded DNA of a host bacterium to modulate transcription of a gene located near a targeted site is provided.
- Contact between the complex of the present invention and double-stranded DNA is carried out by introducing the complex or a nucleic acid encoding the complex into bacteria having a double-stranded DNA of interest (eg, genomic DNA) .
- a double-stranded DNA of interest eg, genomic DNA
- bacteria used in the modification method of the present invention The same bacteria as those used for nucleic acid replication of
- the introduction of the expression vector can be carried out according to the type of bacteria according to known methods (for example, lysozyme method, competent method, PEG method, CaCl 2 coprecipitation method, electroporation method, microinjection method, particle gun method, lipofection method, Agrobacterium method etc.).
- E. coli can be transformed, for example, according to the method described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972) or Gene, 17, 107 (1982).
- Bacillus bacteria can be introduced as a vector according to the method described, for example, in Molecular & General Genetics, 168, 111 (1979).
- the culture of the bacteria into which the vector has been introduced can be carried out according to known methods, depending on the type of bacteria.
- a liquid medium is preferable as a medium used for the culture.
- the medium preferably contains a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of the transformant.
- a carbon source for example, glucose, dextrin, soluble starch, sucrose etc .
- a nitrogen source for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, Inorganic or organic substances such as potato extract liquid
- examples of the inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like.
- yeast extract, vitamins, growth promoting factors and the like may be added to the medium.
- the pH of the culture medium is preferably about 5 to about 8.
- E. coli As a medium for culturing E. coli, for example, M9 medium containing glucose and casamino acid [Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972] is preferable. If necessary, a drug such as, for example, 3 ⁇ -indolylacrylic acid may be added to the medium in order to make the promoter work efficiently.
- Cultivation of E. coli is usually performed at about 15 to about 43 ° C. If necessary, aeration or stirring may be performed. Culturing of the genus Bacillus is usually performed at about 30 to about 40.degree. If necessary, aeration or stirring may be performed.
- PmCDA1 is used as a nucleic acid modifying enzyme
- the present inventors are able to mutate by culturing animal cells and plant cells at a temperature lower than usual (eg, 20 to 26 ° C., preferably about 25 ° C.) It has been confirmed that the introduction efficiency is increased, and also in the case of culturing bacteria, it is preferable to culture at the above-mentioned low temperature.
- RNA introduction of RNA encoding the complex of the present invention into host bacteria can be carried out by the microinjection method, lipofection method or the like. RNA introduction can be repeated once or plural times (for example, 2 to 5 times) at appropriate intervals.
- the present inventors also found that the generation of sequence recognition modules for a plurality of adjacent target nucleotide sequences and their simultaneous use greatly increase the efficiency of mutagenesis compared to targeting a single nucleotide sequence.
- the same effect can be expected from bacteria, which is confirmed using budding yeast.
- the effect is that when a part of both target nucleotide sequences is overlapped, mutagenesis is similarly realized even when both are separated by about 600 bp. It can also occur in both the case where the target nucleotide sequence is in the same orientation (the target strand is the same strand) or in the opposite case where both strands of double stranded DNA are the target strand.
- the method of the present invention demonstrates that mutations can be introduced simultaneously at six sites in bacterial genomic DNA (FIG. 10), and the efficiency of mutagenesis is extremely high. Therefore, in the method for modifying a genome sequence of the present invention or the method for regulating the expression of a target gene, it is possible to target and modify a plurality of DNA regions at completely different positions, or regulate the expression of a plurality of target genes. Therefore, in a preferred embodiment of the present invention, different target nucleotide sequences (which may be in one target gene or in two or more different target genes) may be in the same chromosome or in the same chromosome.
- Two or more nucleic acid sequence recognition modules may be used, each of which specifically binds to a plasmid, or may be located on separate chromosomes or plasmids.
- each one of these nucleic acid sequence recognition modules and a nucleic acid modifying enzyme and / or an inhibitor of base excision repair or a transcriptional regulatory factor form a complex to which a proteolysis tag is added.
- a common one can be used for the nucleic acid modifying enzyme, the inhibitor of base excision repair and the transcriptional regulator.
- RNA-tracrRNA two or more chimeric RNAs can be prepared and used, each of which is a guide RNA that forms a complementary strand with different target nucleotide sequences, and a tracrRNA, respectively.
- each nucleic acid sequence recognition module that specifically binds to a different target nucleotide, a nucleic acid modifying enzyme and / or an inhibitor of base excision repair, Alternatively, transcriptional regulatory factors can be fused.
- an expression vector containing DNA encoding the complex is introduced into host bacteria, but in order to efficiently introduce mutations or In order to regulate the expression of a target gene, it is desirable to maintain the expression of a genome editing complex at a certain level or more for a certain period or more. From this point of view, it is certain that the expression vector is integrated into the host genome, but persistent expression of the genome editing complex increases the risk of off-target cleavage, so that after successful mutagenesis has been achieved. Is preferably removed quickly. Examples of means for removing DNA integrated into the host genome include a method using the Cre-loxP system, a method using a transposon, and the like.
- the target is expressed off-host by transiently expressing the complex of the present invention in the host bacterium for a period of time required for the nucleic acid reaction to occur at the desired time and for the modification of the targeted site to be fixed. Editing of the host genome can be efficiently realized while avoiding the risk of cleavage.
- the time required for the nucleic acid modification reaction to occur and the modification of the targeted site to be fixed varies depending on the type of host bacteria, culture conditions, etc., it is necessary to undergo at least several generations of cell division, About 2-3 days is considered to be necessary.
- a person skilled in the art can appropriately determine a suitable expression induction period based on the culture conditions and the like used.
- the expression induction period of the nucleic acid encoding the complex of the present invention may be extended beyond the above-mentioned "period necessary for the modification of the targeted site to be fixed" as long as the host bacteria cause no side effect. Good.
- a nucleic acid encoding the complex in the mutated CRISPR-Cas system, a DNA encoding a guide RNA, Cas
- a construct expression vector comprising a DNA encoding an effector protein, and optionally a nucleic acid modifying enzyme and / or an inhibitor of base excision repair, or a DNA encoding a transcriptional regulatory factor, in a form capable of controlling the expression period
- transducing in a host is mentioned.
- the “inducible regulatory region” is not particularly limited, and includes, for example, an operon of a temperature sensitive (ts) mutation repressor and an operator controlled thereby.
- ts mutation repressor include, but are not limited to, a ts mutant of cI repressor derived from ⁇ phage, for example. In the case of ⁇ phage cI repressor (ts), it binds to the operator at 30 ° C. or less (eg, 28 ° C.) to suppress downstream gene expression, but at high temperatures of 37 ° C.
- host bacteria into which the nucleic acid encoding the complex of the present invention has been introduced are usually cultured at 30 ° C. or lower, and the temperature is raised to 37 ° C. or higher at an appropriate time, and cultured for a fixed period to perform nucleic acid conversion reaction.
- the mutation is introduced into the target gene, it is possible to minimize the time period during which the expression of the target gene is suppressed by quickly returning to 30 ° C. or less, and when targeting essential genes for the host cell But you can edit efficiently while suppressing the side effects.
- the temperature sensitive mutant of the protein necessary for autonomous replication of the vector can be loaded into the vector containing the DNA encoding the complex of the present invention, As soon as the cell can not replicate autonomously, the vector drops off spontaneously with cell division.
- Such temperature sensitive muteins include, but are not limited to, temperature sensitive mutants of Rep101 ori required for replication of pSC101 ori.
- Rep101 ori acts on pSC101 ori at 30 ° C. or less (eg, 28 ° C.) to allow autonomous replication of the plasmid, but loses function at 37 ° C. or more (eg, 42 ° C.) and the plasmid It can not replicate autonomously. Therefore, in combination with the cI repressor (ts) of ⁇ phage, transient expression of the complex of the present invention and plasmid removal can be performed simultaneously.
- a DNA encoding the complex of the present invention can be used as a host bacterium under the control of an inducible promoter (eg, lac promoter (induced by IPTG), cspA promoter (induced by cold shock), araBAD promoter (induced by arabinose), etc.) And introduce an inducer into the culture medium at an appropriate time (or remove it from the culture medium) to induce expression of the complex, and culture it for a certain period of time to cause a nucleic acid modification reaction etc. to mutate the target gene
- an inducible promoter eg, lac promoter (induced by IPTG), cspA promoter (induced by cold shock), araBAD promoter (induced by arabinose), etc.
- an inducer into the culture medium at an appropriate time (or remove it from the culture medium) to induce expression of the complex, and culture it for a certain period of time to cause a nucleic acid modification reaction etc. to mutate the target gene
- the transient expression of the complex can be realized by stopping the
- pCas9 and p CRISPR plasmids were obtained from the Marraffini laboratory (Non-patent document 8) via Addgene.
- Nickase Cas9 nCas9 (D10A or H840A)
- nuclease deficient Cas9 dCas9 (D10A and H840A) (SEQ ID NOS: 1 and 2) (Jinek, M.
- sgRNA unit (SEQ ID NO: 15) driven by the artificial constitutive promoter J23119 (BBa_J23119 in the registry for standard biological parts) (http://parts.igem.org/Part: BBa_J23119) (SEQ ID NO: 16)
- the plasmid pScI_dCas9-PmCDA1_J23119-sgRNA was amplified by PCR using the primer p346 / p426.
- the sgRNA expression unit contains two BsaI restriction sites for insertion of the target sequence. A pair of oligo DNAs containing the target sgRNA sequence were annealed and ligated to BsaI digested pScI_dCas9-PmCDA1_sgRNA.
- PScI and pScI_dCas9 respectively carry the ⁇ operator only and the operator -dCas9.
- pScI_dCas9-PmCDA1 carries the dCas9-PmCDA1 gene.
- a degradation tag (LVA tag) and UGI gene were added to the C-terminus of dCas9-PmCDA1 gene to obtain plasmids pScI_dCas9-PmCDA1-LVA and pScI_dCas9-PmCDA1-UGI-LVA, respectively.
- the vector plasmid pTAKN-2 has the pMB1 origin of replication corresponding to pSC101.
- the sgRNA unit with promoter J23119 was excised from the synthetic oligonucleotide using EcoRI-HindIII and ligated into the cloning vector pTAKN2.
- golden gate assembly of PCR products using BsaI digestion-ligation (Engler, C.
- Three target sequences in tandem (pSBP804, galK_10-galK_11-gal_13; pSBP806 , GalK_2-xylB_1-manA_1; pSBP808, pta_1-adhE_3-tpiA_2) was constructed.
- ⁇ Mutagenesis assay Chemically transformed DH5 ⁇ or BW25113 cells with the plasmid of interest in 1 mL of SOC medium (2% Bacto Tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO 4 and 20 mM glucose) It precultured. After incubating at 28 ° C. for 2-3 hours, the cell culture is diluted 1:10 with 1 ml of Luria-Bertani (LB) medium or terrific broth (TB), optionally with antibiotics (chloramphenicol ( 25 ⁇ g / ml) and / or kanamycin (30 ⁇ g / ml) were supplemented.
- SOC medium 2% Bacto Tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO 4 and 20 mM glucose
- M63 minimal medium (2 g / L (NH 4 ) 2 SO 4 , 13.6 g / L KH 2 PO 4 containing 0.2% glycerol and 2-deoxy-galctose (2-DOG) for positive selection of galK gene disruption , 0.5 mg / L FeSO 4 -7H 2 O, 1 mM MgSO 4 , 0.1 mM CaCl 2 and 10 ⁇ g / ml thiamine (Warming, S. et al., Nucleic Acids Res. 33, 1-12 (2005). ), Cells were allowed to grow.
- rifampicin resistant mutations of the rpoB gene cells were grown in LB medium containing 50 ⁇ g / ml rifampicin. For sequence analysis, colonies were randomly picked, amplified directly by PCR using appropriate primers, and analyzed by Sanger method using 3130 XL Genetic Analyzer (Applied Biosystems). t-test statistical analysis was performed using Excel software (Microsoft).
- BW25113 cells with each expression construct (dCas9, dCas9-PmCDA1, dCas9-PmCDA1-LVA-UGI, and dCas9-PmCDA1 with rpoB_1 target) are precultured overnight and diluted 1:10 in 1 mL LB medium The mice were grown for 6 hours at 37 ° C. for induction and then incubated overnight at 28 ° C. The cells were spread on plate media containing rifampicin to isolate single colonies. Three independent colonies each were inoculated into TB medium.
- Genomic DNA is extracted using Wizard Genomic DNA Purification Kit (Promega) and then ultrasonically fragmented using Bioruptor UCD-200 TS Sonication System (Diagenote) to obtain fragments with a size distribution of 500 to 1000 bp
- the A genomic DNA library was prepared using NEBNext Ultra DNA Library Prep Kit from Illumina (New England Biolabs) and labeled with Dual Index Primer. Library size selection was performed using Agencourt AMPure XP (Beckman Coulter) to obtain labeled fragments ranging in length from 600-800 bp. Size distribution was evaluated by Agilent 2100 Bioanalyzer system (Agilent Technologies).
- DNA was quantified using a Qibit HS dsDNA HS Assay Kit and a fluorometer (Thermo Fisher Scientific). Sequencing was performed using the MiSeq Sequencing System (Illumina) and MiSeq Reagent Kit v3 to obtain a read length of 2 ⁇ 300 bp where coverage of about 20 times the genome size can be expected. Data analysis was performed using CLC Genomic Workbench 9.0 (CLC bio). Sequenced reads were paired to merge duplicate reads in the read pair and trimmed based on a quality limit of 0.01 with an ambiguity of up to 2.
- ⁇ Deep sequencing> Incubate DH5 ⁇ cells expressing dCas9-PmCDA1 or dCas9-PmCDA1-UGI-LVA with gRNA targeting the galK, gsiA, ycbF or yfiH gene overnight, dilute 1:10 in 1 mL LB medium and induce For 6 hours at 37.degree. Cell cultures were harvested and genomic DNA was extracted. A fragment ( ⁇ 0.3 kb) containing the target region was directly amplified from the extracted genomic DNA using primer pairs (p 685 to p 696). Amplicons were labeled with Dual Index Primer. An average of over 30,000 reads per sample were analyzed with the MiSeq sequencing system.
- Sequence reads were paired and trimmed based on a quality limit of 0.01 with a maximum of 2 ambiguities to merge duplicate reads in the lead pair.
- Example 1 Deaminase-Mediated Target Mutagenesis in E. coli
- a temperature-inducible ⁇ operator system Wang, Y. et al. , Nucleic Acids Res. 40, (2012).
- nCas (H840) -CDA showed high transformation efficiency similar to dCas and dCas-CDA, and was shown to be advantageous for cell growth and cell survival.
- the galactose analogue 2-deoxy-D-galactose (2-DOG) then targets the galK gene, which can actively select for loss of function, to quantitatively assess the efficiency of target mutagenesis. Used as 2-DOG is catalyzed by galactokinase of the galK gene product and becomes a harmful compound (Warming, S. et al., Nucleic Acids Res. 33, 1-12 (2005).).
- Target-AID induces a mutation at a cytosine nucleotide (C) located about 15 to 20 bases in the core region of the upstream 16 to 19 bases of the protospacer flanking motif (PAM) sequence (Non-patent document 11) It is known (non-patent document 11) (Fig. 1 (a)).
- C cytosine nucleotide
- PAM protospacer flanking motif
- rpoB an essential gene encoding the ⁇ -subunit of RNA polymerase. While inhibition of rpoB gene function causes suppression of cell proliferation and cell death, certain point mutations in the rpoB gene are known to confer rifampicin resistance (Jin, D. J. et al., J. Mol. Mol. Biol. 202, 245-253 (1988).). The target sequence was designed to induce a point mutation that confers rifampicin resistance (FIG. 5 (a)). Without apparent growth inhibition, transformed cells acquired rifampicin resistance at a frequency of almost 100% (FIG. 4 (b)).
- Example 2 Effects of sgRNA Length and Uracil DNA Glycosylase Inhibitor on the Frequency and Location of Mutations
- sgRNA Length and Uracil DNA Glycosylase Inhibitor were used to comprehensively analyze the mutation efficiency and location of deep sequencing analysis using 18 target sequences of galK gene ( Figures 6 and 7). Seven targets showed high efficiency (61.7-95.1%) of mutagenesis while 5 showed low (1.4-9.2%) mutagenesis. The most effective mutation position is 17-20 bases upstream of PAM, which is consistent with previous studies in higher organisms.
- the mutation frequency is also the length of the target sequence Changed depending on the
- uracil DNA glycosylase inhibitors from bacteriophage PBS2 (Zhigang, W. et al., Gene 99, 31-37 (1991).) And proteolytic tags (LVA tag) (LVA tag) Andersen, J. B. et al., Appl. Environ. Microbiol. 64, 2240-2246 (1998).) was introduced by fusing to the C-terminus of dCas-CDA. UGI promotes mutagenesis by cytidine deamination to inhibit removal of uracil (the direct product of cytosine deamination) from DNA (10, 11).
- LVA tag protects the cells from injury and suppresses the emergence of escaper cells by reducing the half-life of the dCas-CDA-UGI protein which may be potentially harmful if overexpressed There is expected.
- whole genome sequence analysis was performed on cells expressing dCas, dCas-CDA and dCas-CDA-UGI-LVA. While dCas-CDA induced 0-2 SNV mutations, dCas-CDA-UGI-LVA induced 21-30 mutations with no positional bias across the genome (Tables 3 and 4).
- Rifampicin selection clones expressing each construct (dCas, dCas-CDA or dCas-CDA-LVA-UGI) not using sgRNA were whole genome sequenced.
- the biological triplicates of dCas-CDA and dCas-CDA-LVA-UGI are shown.
- Sequence coverage was calculated as the sum of base pairs mapped to sequences spanning 4,631 Mbp of E. coli BW 25 113 genomic sequence. The list of unique mutations is shown in Table 4.
- dCas-CDA-UGI-LVA shows strong mutagenesis at all target sites regardless of the length and position of the target sequence ( Figure 6, right bar), showing mutation spectra using sgRNAs of different lengths It became possible to compare. As a result, it was shown that in galK_9 and galK_11, the mutation spectrum was expanded toward the 5 'end (FIG. 6). To further characterize the effect of sgRNA on the length of the target sequence, C-rich target sequences of 18 nt, 20 nt, 22 nt and 24 nt in length were tested ( Figure 8 (a) and (b)). The mutational spectra for each of the five target sites consistently showed peak shift towards the 5 'end and expansion of the window as the target sequence became longer (Fig. 8 (c)).
- Example 3 Multiplex Mutagenesis
- tandem repeats of the sgRNA expression unit were assembled on a plasmid separate from the modifying plasmid. Construct plasmids targeting three sites (galK_10, galK_11 and galK_13) of galK gene, and have modified vector expressing dCas, dCas-CDA, dCas-CDA-LVA or dCas-CDA1-UGI-LVA
- the cells were co-introduced.
- nonspecific mutagenesis effects were evaluated by analyzing the occurrence of rifampicin resistant mutations (FIG. 9).
- dCas-CDA showed about 10-fold increase over background mutation frequency
- dCas-CDA-UGI-LVA showed a further 10-fold increase over dCas-CDA mutation frequency.
- both dCas-CDA and dCas-CDA-LVA were not efficient enough at the same time to obtain triple mutants, one mutation occurred at both, and at least the target mutation rate was significant depending on the presence or absence of LVA. There was no significant difference. Therefore, it was shown that addition of LVA can suppress nonspecific mutagenesis while maintaining mutational efficiency.
- the mutation frequency was lower when compared with the result of each single target that produced 100% (8/8) for each target (FIG.
- dCas-CDA-UGI-LVA succeeded in inducing a triple mutation in 5 out of 8 analyzed clones (Fig. 9 (b) and (d)). Therefore, it was shown that combining UGI and LVA can suppress nonspecific mutagenesis while achieving high mutation efficiency.
- Target-AID can edit multiple loci at once using the same sgRNA sequence without inducing genomic instability.
- four major transposable elements TE: IS1, 2, 3 and 5
- TE transposable elements
- the sgRNA was designed to contain the consensus sequence of each TE transposase gene and introduced a stop codon (FIG. 11).
- coli Top10 cells were transformed with two plasmids that respectively express dCas-CDA-UGI-LVA and four target sgRNAs. Procedures for isolation and verification of IS-edited cells are shown in FIG. 12 and described in detail below. After double transformation and selection, colonies were amplified by PCR and first sequenced at sites IS5-1, IS5-2, IS5-11, IS5-12. The IS5 target proved to be inefficient. Of the four colonies analyzed, one contained three mutation sites and one heterologous genotype site (IS5-1). The cells were then suspended in liquid medium, spread on plates and colonies reisolated.
- IS5-1 Three of the eight colonies contained mutations in IS5-1, and for two of them, the sequences of the remaining 24 IS loci were further sequenced, including all mutated sites, but one It was shown to contain an imperfect, heterologous genotype site (IS5-5).
- the cells were then suspended and expanded to obtain 4 out of 6 reisolated clones containing mutations in IS5-5.
- One of the clones was sequenced at the IS5 site and found to contain one heterologous genotype site (IS5-2).
- Eight clones were isolated again, and six clones contained the IS5-2 mutation. Two clones were spread on non selective media to obtain cells that had lost the plasmid.
- Region indicates the target site in the DH10B database.
- Strand indicates the orientation of the target sequence.
- the expected off target sequence was determined as described herein.
- the mismatch indicates the number of mismatches between the on target sequence and the off target sequence. Mismatched nucleotides are highlighted in bold. The frequency of C to T mutation in each sequence highlighted by gray box is shown.
- Example 5 Comparison of transformation efficiency of E. coli with a yeast expression vector LbCpf1 (SEQ ID NO: 326 and 327) as a Cas effector protein, YAASV and YALAA as a protein degradation tag Yeast expression vector (vector 3685: Cpf1-NLS) -3xFlag-YAASV (SEQ ID NO: 328), vector 3687: Cpf1-NLS-3xFlag-YALAA (SEQ ID NO: 329), and a control vector having no nucleic acid encoding a proteolysis tag (vector 3687: Cpf1-NLS-3xFlag) (SEQ ID NO: 330)) was made based on the pRS315 vector.
- FIG. 13 shows a schematic view of each vector.
- the DNA solution containing each vector was adjusted to 2 ng / ⁇ l, and 1 ⁇ l (2 ng) of the DNA solution was added to 20 ⁇ l of E. coli Top 10 competent cells for transformation. Thereafter, 200 ⁇ l of SOC was added, and the cells were recovered and cultured at 37 ° C. for 1 hour, and growth was stopped on ice for 5 minutes, and then 1 ⁇ l of 50 mg / ml Amp was added. A portion of the culture (1 ⁇ l and 10 ⁇ l) was diluted with TE, applied to an LB + Amp plate, cultured overnight at 37 ° C., and the number of colonies was counted. The results are shown in Table 6.
- the present invention provides a low-toxic vector that can be stably amplified even in host bacteria and a genome editing complex encoded by the vector. According to the method of genome editing using the vector and the nucleic acid modifying enzyme of the present invention, it becomes possible to modify the gene of host bacteria while suppressing nonspecific mutations and the like. This method is applicable to a wide range of bacteria and is extremely useful because it does not rely on host dependent factors such as RecA.
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Abstract
Description
[1] 二本鎖DNA中の標的ヌクレオチド配列と特異的に結合する核酸配列認識モジュールと、(i)疎水性アミノ酸3残基をC末端に含むペプチド、又は(ii)該アミノ酸残基の少なくとも一部がセリンに置換されたアミノ酸3残基をC末端に含むペプチドからなるタンパク質分解タグとが結合した、複合体。
[2] 前記複合体が、核酸改変酵素がさらに結合した複合体であって、標的化された部位の1以上のヌクレオチドを他の1以上のヌクレオチドに変換し又は欠失させ、あるいは該標的化された部位に1以上のヌクレオチドを挿入する、[1]に記載の複合体。
[3] 前記アミノ酸3残基が、ロイシン-バリン-アラニン、ロイシン-アラニン-アラニン、アラニン-アラニン-バリン又はアラニン-セリン-バリンである、[1]又は[2]に記載の複合体。
[4] 前記核酸配列認識モジュールが、Casの2つのDNA切断能のうちの一方のみ、又は両方のDNA切断能が失活したCRISPR-Casシステムである、[1]~[3]のいずれかに記載の複合体。
[5] 前記複合体が、CRISPR-Casシステムと、タンパク質分解タグとが結合した複合体である、[1]~[3]のいずれかに記載の複合体。
[6] 前記核酸改変酵素が核酸塩基変換酵素又はDNAグリコシラーゼである、[2]~[4]のいずれかに記載の複合体。
[7] 前記核酸塩基変換酵素がデアミナーゼである、[6]に記載の複合体。
[8] 塩基除去修復のインヒビターがさらに結合した、[6]又は[7]に記載の複合体。
[9] [1]~[8]のいずれかに記載の複合体をコードする核酸。
[10] 細菌の二本鎖DNAの標的化された部位を改変する、又は該部位の近傍で二本鎖DNAにコードされる遺伝子の発現を調節する方法であって、選択された二本鎖DNA中の標的ヌクレオチド配列と特異的に結合する核酸配列認識モジュールと、(i)疎水性アミノ酸3残基をC末端に含むペプチド、又は(ii)該アミノ酸残基の少なくとも一部がセリンに置換されたアミノ酸3残基をC末端に含むペプチドからなるタンパク質分解タグとが結合した複合体を、該二本鎖DNAと接触させる工程を含む、方法。
[11] 前記複合体が、核酸改変酵素がさらに結合した複合体であって、該標的化された部位の1以上のヌクレオチドを他の1以上のヌクレオチドに変換する又は欠失させる、あるいは該標的化された部位に1以上のヌクレオチドを挿入する工程を含む、[10]に記載の方法。
[12] 前記アミノ酸3残基が、ロイシン-バリン-アラニン、ロイシン-アラニン-アラニン、アラニン-アラニン-バリン又はアラニン-セリン-バリンである、[10]又は[11]に記載の方法。
[13] 前記核酸配列認識モジュールが、Casの2つのDNA切断能のうちの一方のみ、又は両方のDNA切断能が失活したCRISPR-Casシステムである、[10]~[12]のいずれかに記載の方法。
[14] 前記複合体が、CRISPR-Casシステムと、タンパク質分解タグとが結合した複合体である、[10]~[12]のいずれかに記載の方法。
[15] 異なる標的ヌクレオチド配列とそれぞれ特異的に結合する、2種以上の核酸配列認識モジュールを用いることを特徴とする、[10]~[14]のいずれかに記載の方法。
[16] 前記異なる標的ヌクレオチド配列が、異なる遺伝子内に存在する、[15]に記載の方法。
[17] 前記核酸改変酵素が核酸塩基変換酵素又はDNAグリコシラーゼである、[10]~[13]、[15]及び[16]のいずれかに記載の方法。
[18] 前記核酸塩基変換酵素がデアミナーゼである、[17]に記載の方法。
[19] 該複合体が、塩基除去修復のインヒビターがさらに結合したものである、[17]又は[18]に記載の方法。
[20] 二本鎖DNAと複合体との接触が、該二本鎖DNAを有する細菌への、該複合体をコードする核酸の導入により行われる、[10]~[19]のいずれかに記載の方法。
本発明は、二本鎖DNA中の標的ヌクレオチド配列と特異的に結合する核酸配列認識モジュールと、タンパク質分解タグとが結合したゲノム編集用複合体及び該複合体をコードする核酸を提供する。本発明のゲノム編集用複合体の一態様において、核酸改変酵素がさらに結合した複合体(即ち、核酸配列認識モジュールと、核酸改変酵素と、タンパク質分解タグとが結合した複合体)であって、標的化された部位の核酸を改変し得る複合体を提供する。一態様において、二本鎖DNAの改変効率を向上させるため、該複合体には、塩基除去修復のインヒビターがさらに結合していてもよい。また、本発明のゲノム編集用複合体の別の態様において、核酸配列認識モジュールと、タンパク質分解タグとが少なくとも結合した複合体であって、標的化された部位の近傍で二本鎖DNAにコードされる遺伝子の発現を調節し得る複合体を提供する。一態様において、該複合体には、転写調節因子がさらに結合されていてもよい。以下では、核酸改変酵素、塩基除去修復のインヒビター及び転写調節因子の少なくともいずれかが結合した複合体と、いずれも結合していない複合体とをまとめて、「本発明の複合体」又は「ゲノム編集用複合体」と称することがあり、核酸改変酵素が結合した複合体を、特に「核酸改変酵素複合体」と称することがある。また、これらの複合体をコードする核酸をまとめて、「本発明の核酸」と称することがある。
核酸改変酵素及び塩基除去修復のインヒビターをコードするDNAも、同様に、使用する酵素のcDNA配列情報をもとにオリゴDNAプライマーを合成し、当該酵素を産生する細胞より調製した全RNAもしくはmRNA画分を鋳型として用い、RT-PCR法によって増幅することにより、クローニングすることができる。例えば、PBS2由来のUGIをコードするDNAは、NCBI/GenBankデータベースに登録されているDNA配列(accession No. J04434)をもとに、CDSの上流及び下流に対して適当なプライマーを設計し、PBS2由来mRNAからRT-PCR法によりクローニングできる。
クローン化されたDNAは、そのまま、又は所望により制限酵素で消化するか、適当なリンカー(例、GSリンカー、GGGARリンカー等)、スペーサー(例、FLAG配列等)及び/又は核移行シグナル(NLS)(目的の二本鎖DNAがミトコンドリアや葉緑体DNAの場合は、各オルガネラ移行シグナル)を付加して、タンパク質をコードするDNAを調製することができる。また、さらに核酸配列認識モジュールをコードするDNAとライゲーションして、融合タンパク質をコードするDNAを調製することができる。
発現ベクターとしては、大腸菌由来のプラスミド(例、pBR322,pBR325,pUC12,pUC13);枯草菌由来のプラスミド(例、pUB110,pTP5,pC194);酵母由来プラスミド(例、pSH19,pSH15);昆虫細胞発現プラスミド(例:pFast-Bac);動物細胞発現プラスミド(例:pA1-11、pXT1、pRc/CMV、pRc/RSV、pcDNAI/Neo);λファージなどのバクテリオファージ;バキュロウイルスなどの昆虫ウイルスベクター(例:BmNPV、AcNPV);レトロウイルス、ワクシニアウイルス、アデノウイルスなどの動物ウイルスベクターなどが用いられる。
プロモーターとしては、遺伝子の発現に用いる宿主に対応して適切なプロモーターであればいかなるものでもよい。核酸改変酵素として核酸分解酵素を用いる場合には、毒性のために宿主細胞の生存率が著しく低下する場合があるので、誘導プロモーターを使用して誘導開始までに細胞数を増やしておくことが望ましい。一方で、核酸改変酵素として核酸塩基変換酵素及びDNAグリコシラーゼを用いる場合、あるいは核酸改変酵素を用いない場合には、本発明の複合体を発現させても十分な細胞増殖が得られるので、構成プロモーターも制限なく使用することができる。
例えば、宿主が動物細胞である場合、SRαプロモーター、SV40プロモーター、LTRプロモーター、CMV(サイトメガロウイルス)プロモーター、RSV(ラウス肉腫ウイルス)プロモーター、MoMuLV(モロニーマウス白血病ウイルス)LTR、HSV-TK(単純ヘルペスウイルスチミジンキナーゼ)プロモーターなどが用いられる。なかでも、CMVプロモーター、SRαプロモーターなどが好ましい。
宿主が大腸菌である場合、J23シリーズのプロモーター(例:J23119プロモーター)、trpプロモーター、lacプロモーター、recAプロモーター、λPLプロモーター、lppプロモーター、T7プロモーターなどが好ましい。
宿主がバチルス属菌である場合、SPO1プロモーター、SPO2プロモーター、penPプロモーターなどが好ましい。
宿主が酵母である場合、Gal1/10プロモーター、PHO5プロモーター、PGKプロモーター、GAPプロモーター、ADHプロモーターなどが好ましい。
宿主が昆虫細胞である場合、ポリヘドリンプロモーター、P10プロモーターなどが好ましい。
宿主が植物細胞である場合、CaMV35Sプロモーター、CaMV19Sプロモーター、NOSプロモーターなどが好ましい。
エシェリヒア属菌としては、例えば、エシェリヒア・コリ(Escherichia coli)K12・DH1〔Proc. Natl. Acad. Sci. USA,60,160 (1968)〕,エシェリヒア・コリJM103〔Nucleic Acids Research,9,309 (1981)〕,エシェリヒア・コリJA221〔Journal of Molecular Biology,120,517 (1978)〕,エシェリヒア・コリHB101〔Journal of Molecular Biology,41,459 (1969)〕,エシェリヒア・コリC600〔Genetics,39,440 (1954)〕、エシェリヒア・コリDH5α、エシェリヒア・コリBW25113などが用いられる。
バチルス属菌としては、例えば、バチルス・サブチルス(Bacillus subtilis)MI114〔Gene,24,255 (1983)〕,バチルス・サブチルス207-21〔Journal of Biochemistry,95,87 (1984)〕などが用いられる。
これに対し、CRISPR-Casシステムは、標的ヌクレオチド配列に対して相補的なガイドRNAにより目的の二本鎖DNAの配列を認識するので、標的ヌクレオチド配列と特異的にハイブリッド形成し得るオリゴDNAを合成するだけで、任意の配列を標的化することができる。
従って、本発明のより好ましい実施態様においては、核酸配列認識モジュールとして、DNA切断能の両方の活性が維持されたCRISPR-Casシステム、あるいはCasの1つのみ、又は両方のDNA切断能が失活したCRISPR-Casシステム(CRISPR-変異Cas)が用いられる。
あるいはCasエフェクタータンパク質をコードするDNAは、核酸配列認識モジュールをコードするDNAやDNAグリコシラーゼをコードするDNAについて上記したのと同様の方法により、化学合成又はPCR法もしくはGibson Assembly法との組み合わせで、用いる宿主細胞での発現に適したコドン使用を有するDNAとして構築することもできる。
ここで「標的鎖」とは、標的ヌクレオチド配列のcrRNAとハイブリッド形成する方の鎖を意味し、その反対鎖で標的鎖とcrRNAとのハイブリッド形成により一本鎖状になる鎖を「非標的鎖(non-targeted strand)」と呼ぶこととする。また、核酸塩基変換反応は通常、一本鎖状になった非標的鎖上で起こる場合が多いと推定されるので、標的ヌクレオチド配列を片方の鎖で表現する場合(例えばPAM配列を表記する場合や、標的ヌクレオチド配列とPAMとの位置関係を表す場合等)、非標的鎖の配列で代表させるものとする。
1.に記載した本発明の複合体や核酸を、宿主、特に細菌に導入し、当該宿主を培養することによって、宿主の二本鎖DNAの標的化された部位を改変する、あるいは標的化された部位の近傍で二本鎖DNAにコードされる遺伝子の発現を調節することができる。従って、別の実施態様において、核酸改変酵素複合体を、宿主細菌の二本鎖DNAと接触させ、標的化された部位の1以上のヌクレオチドを他の1以上のヌクレオチドに変換する又は欠失させる、あるいは該標的化された部位に1以上のヌクレオチドを挿入する工程を含む、細菌の二本鎖DNAの標的化された部位を改変する方法(以下「本発明の改変方法」ともいう)が提供される。核酸改変酵素として核酸塩基変換酵素又はDNAグリコシラーゼを用いることで、標的化された部位において、二本鎖DNAの少なくとも一方の鎖を切断することなく、該標的化された部位を改変することができる。また、さらに別の実施態様において、本発明の複合体を、宿主細菌の二本鎖DNAと接触させ、標的化された部位の近傍に位置する遺伝子の転写を調節する方法が提供される。
大腸菌は、例えば、Proc. Natl. Acad. Sci. USA,69,2110 (1972)やGene,17,107 (1982)などに記載の方法に従って形質転換することができる。
バチルス属菌は、例えば、Molecular & General Genetics,168,111 (1979)などに記載の方法に従ってベクター導入することができる。
バチルス属菌の培養は、通常約30~約40℃で行なわれる。必要により、通気や撹拌を行ってもよい。
また、本発明者らは、核酸改変酵素としてPmCDA1を用いる場合に、動物細胞や植物細胞を通常よりも低温(例えば、20~26℃、好ましくは、約25℃)で培養することにより、変異導入効率を上昇させることを確認しており、細菌を培養する場合も、上記低温で培養することも好ましい。
温度感受性変異を利用する場合、例えば、ベクターの自律複製に必要なタンパク質の温度感受性変異体を、本発明の複合体をコードするDNAを含むベクターに搭載することにより、該複合体の発現後、速やかに自律複製が出来なくなり、細胞分裂に伴って該ベクターは自然に脱落する。このような温度感受性変異タンパク質としては、pSC101 oriの複製に必要なRep101 oriの温度感受性変異体が挙げられるが、これに限定されない。Rep101 ori (ts)は30℃以下(例、28℃)では、pSC101 oriに作用してプラスミドの自律複製を可能にするが、37℃以上(例、42℃)になると機能を失い、プラスミドは自律複製できなくなる。従って、上記λファージのcIリプレッサー(ts)と併用することで、本発明の複合体の一過的発現と、プラスミド除去とを、同時に行うことができる。
<菌株、プラスミド、プライマー及び標的化gRNAデザイン>
大腸菌株DH5α((F- endA1 supE44 thi-1 recA1 relA1 gyrA96 deoR phoA Φ80dlacZ ΔM15 Δ(lacZYA-argF)U169, hsdR17 (rK-, mK+), λ- )(TaKaRa-Bio)、BW25113(lacI+ rrnBT14 ΔlacZWJ16hsdR514 ΔaraBADAH33 ΔrhaBADLD78 rph-1 Δ(araB-D)567 Δ(rhaD-B)568ΔlacZ4787(::rrnB-3) hsdR514 rph-1)及びTop10(F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-)(Invitrogen)を使用した。実施例で用いたプラスミド及びプライマーを、それぞれ表1及び表2に挙げた。標的化gRNAベクターを構築するためのオリゴDNAのペアを、次のように設計した:5'-tagc-(標的配列)-3 '及び5'-aaac-(標的配列の逆相補的配列)-3'。
pCas9及びpCRISPRプラスミドを、Addgeneを介して、Marraffini研究室(非特許文献8)から入手した。Nickase Cas9:nCas9(D10A又はH840A)及びヌクレアーゼ欠損Cas9:dCas9(D10A及びH840A)(配列番号1及び2)(Jinek, M. et al., Science 337, 816-822 (2012).)をPCR法により作製した。 PmCDA1(配列番号3及び4)は、121アミノ酸のペプチドリンカー(配列番号5及び6)を用いて、nCas9又はdCas9のC末端に融合した(図1)。
目的のプラスミドで化学的に形質転換したDH5α又はBW25113細胞を、1 mLのSOC培地(2% Bacto Tryptone、0.5%酵母エキス、10 mM NaCl、2.5mM KCl、1 mM MgSO4及び20 mMグルコース)で前培養した。28℃で2~3時間インキュベートした後、細胞培養物を1 mlのルリア-ベルターニ(LB)培地又はterrific broth(TB)で1:10希釈し、必要に応じて抗生物質(クロラムフェニコール(25 μg/ml)及び/又はカナマイシン(30 μg/ml))を補充した。マキシマイザ(TAITEC)を用いて、28℃、100K rpmで一晩増殖させた。翌日、細胞培養物を再び1 ml培地で1:10に希釈し、誘導のために37℃で6時間培養し、28℃で一晩インキュベートした。次いで、細胞培養物を適切な抗生物質を補充したLB又はTB寒天プレート上に連続希釈してスポットし、28℃で一晩インキュベートして単一のコロニーを形成させた。
各発現コンストラクト(dCas9、dCas9-PmCDA1、dCas9-PmCDA1-LVA-UGI、及びrpoB_1標的を有するdCas9-PmCDA1)を有するBW25113細胞を一晩前培養し、1 mLのLB培地で1:10に希釈し、誘導のために37℃で6時間増殖させ、その後28℃で一晩インキュベートした。細胞をリファンピシンを含有するプレート培地上に広げて、単一のコロニーを分離した。それぞれ3個の独立したコロニーをTB培地に接種した。Wizard Genomic DNA Purification Kit(Promega)を用いて、ゲノムDNAを抽出し、次いでBioruptor UCD-200 TS Sonication System(Diagenote)を用いて超音波により断片化し、500~1000 bpのサイズ分布を有する断片を得た。ゲノムDNAライブラリーを、Illumina(New England Biolabs)のNEBNext Ultra DNA Library Prep Kitを用いて調製し、Dual Index Primerで標識した。ライブラリーのサイズ選択を、Agencourt AMPure XP(Beckman Coulter)を用いて行い、600~800bpの範囲の長さの標識断片を得た。サイズ分布を、Agilent 2100 Bioanalyzer system(Agilent Technologies)により評価した。Qibit HS dsDNA HS Assay Kit及び蛍光光度計(Thermo Fisher Scientific)を用いてDNAを定量した。ゲノムサイズの約20倍のカバレッジが期待できる、2×300bpのリード長を得るため、シーケンシングを、MiSeqシーケンシングシステム(Illumina)及びMiSeq Reagent Kit v3を用いて行った。データ解析は、CLC Genomic Workbench 9.0を用いて行った(CLC bio)。シーケンスリードを対にして、リードペア内の重複するリードをマージし、最大2のアンビギュイティで、0.01の品質限界に基づいてトリミングした。次の設定(Masking mode = no masking, Mismatch cost = 2, Insertion cost = 3, Deletion cost = 3, Length fraction = 0.5, Similarity fraction = 0.8, Global alignment = No, Auto-detect paired distances = Yes, Nonspecific match handling = ignore)で、リードをE.coli BW25113の参照ゲノムにマッピングした。ローカルリアライメントを、デフォルト設定で行った(Realign unaligned ends = Yes, Multi-pass realignment = 2)。バリアントコーリングを、次の設定(Ignore positions with coverage = 1,000,000, Ignore broken pairs = Yes, Ignore Nonspecific matches = Reads, Minimum coverage = 5, Minimum count = 2, Minimum frequency = 50%, Base quality filter = No, Read detection filter = No, Relative read direction filter = Yes, Significance = 1%, Read position filter = No, Remove pyro-error variants = No)で行った。出力ファイルを、Excel(Microsoft)を用いて並べ替えた。
galK、gsiA、ycbF又はyfiH遺伝子を標的とするgRNAと共にdCas9-PmCDA1又はdCas9-PmCDA1-UGI-LVAを発現するDH5α細胞を一晩インキュベートし、1 mLのLB培地で1:10に希釈し、誘導のために37℃で6時間増殖させた。細胞培養物を収集し、ゲノムDNAを抽出した。抽出されたゲノムDNAからプライマーペア(p685~p696)を用いて、標的領域を含む断片(~0.3kb)を直接増幅した。アンプリコンをDual Index Primerで標識した。サンプル当たり平均30,000以上のリードを、MiSeqシーケンシングシステムで解析した。シーケンスリードを対にして、最大2のアンビギュイティで、0.01の品質限界に基づいてトリミングし、リードペア内の重複するリードをマージした。次の設定(Masking mode = no masking, Mismatch cost = 2, Insertion cost = 3, Deletion cost = 3, Length fraction = 0.5, Similarity fraction = 0.8, Global alignment = No, Auto-detect paired distances = Yes, Nonspecific match handling = Map randomly)で、リードをそれぞれ参照配列にマッピングした。出力ファイルを、Excelを用いて並べ替えた。
デアミナーゼに媒介される標的の変異誘発が細菌に適用できるかどうかを評価するために、温度誘導性λオペレーターシステム(Wang, Y. et al., Nucleic Acids Res. 40, (2012).)下で、P.marinus(ウミヤツメ)由来のシトシンデアミナーゼPmCDA1(非特許文献11)に融合した触媒的に不活性なCas9(dCas:D10A及びH840Aの変異)を発現し、人工の構成的プロモーター J23119(図1(b))の下で、20ヌクレオチド(nt)の標的配列-gRNA足場のハイブリッド(sgRNA)下でCDAを発現する、細菌を標的としたAID(Target-AID)ベクターを構築した。真核生物では、より高い変異効率を達成するため、デアミナーゼと組み合わせたニッカーゼCas9(nCas:D10A変異)を使用することができるが(非特許文献10、11)、nCas(D10A)-CDAを発現するプラスミドでは、形質転換効率が低いことが示された。このことは、大腸菌において、完全なCas9ヌクレアーゼと同様に、nCas(D10A)-CDAが重篤な細胞増殖及び/又は細胞死を引き起こすことを示唆する(図2)。一方、nCas (H840)-CDAは、dCasやdCas-CDAと同様に高い形質転換効率を示し、細胞増殖や細胞の生存にとって有利であることが示された。
次に標的の変異誘発の効率を定量的に評価するために、ガラクトースのアナログ 2-デオキシ-D-ガラクトース(2-DOG)により、機能の喪失を積極的に選択することができるgalK遺伝子を標的として用いた。2-DOGは、galK遺伝子産物のガラクトキナーゼによって触媒され、有害な化合物となる(Warming, S. et al., Nucleic Acids Res. 33, 1-12 (2005).)。Target-AIDは、プロトスペーサー隣接モチーフ(PAM)配列(非特許文献11)の上流16~19塩基のコア領域内の約15~20塩基目に位置するシトシンヌクレオチド(C)において、変異を誘導することが知られている(非特許文献11)(図1(a))。galK遺伝子において、ストップコドンが導入されるように標的の配列(図3)を選択したところ、2-DOGに対してほぼ100%の生存率を誘導し、このことは非常に効率的な変異誘発を示唆した(図4(a))。2-DOGを含まない培地で培養した細胞についてシーケンシング分析を行った場合、8個のコロニーのうち6個が予想通り変異していた。予想通り-17位及び/又は20位で、CからTへの置換が観察された。
変異効率と位置を総合的に分析するために、galK遺伝子の18個の標的配列を用いてディープシーケンシング分析を行った(図6、図7)。7個の標的は高い効率(61.7~95.1%)の変異誘発を示したが、一方で5個は低い(1.4~9.2%)変異誘発を示した。最も効果的な変異位置はPAMの上流17-20塩基であり、これは高等生物における以前の研究と一致した。より長い標的配列を有するsgRNAがgalK_8及びgalK_13でより高い効率を示し、galK_9及びgalK_11でより低い効率を示したことから理解できるように(図6、左側のバー)、変異頻度も標的配列の長さに依存して変化した。
多重編集のために、sgRNA発現ユニットのタンデムなリピートを、改変用プラスミドとは別のプラスミド上に組み立てた。galK遺伝子の3箇所の部位(galK_10、galK_11及びgalK_13)を標的とするプラスミドを構築し、dCas、dCas-CDA、dCas-CDA-LVA又はdCas-CDA1-UGI-LVAを発現する改変用ベクターを有する細胞に同時導入した。まず、リファンピシン耐性変異の発生を分析することにより、非特異的な変異誘発効果を評価した(図9)。dCas-CDAはバックグラウンドの変異頻度より約10倍の増加を示したが、dCas-CDA-UGI-LVAは、dCas-CDAの変異頻度よりさらに10倍の増加を示した。dCas-CDA及びdCas-CDA-LVAでは、同時に三重変異体を得るのに十分な効率ではなかったが、1箇所の変異はどちらでも生じており、少なくとも標的の変異率は、LVAの有無によって有意な差は認められなかった。従って、LVAを付加することで、変異効率を維持しつつ非特異的な変異誘発を抑制することができることが示された。また、dCas-CDA-UGI-LVAでは、各標的に対して100%(8/8)を生じたそれぞれの単一標的の結果と比較した場合には、変異頻度は低かったものの(図9(c)及び(d))、dCas-CDA-UGI-LVAでは、解析した8クローンのうち5クローンで3重変異を誘発することに成功した(図9(b)及び(d))。従って、UGIとLVAを組み合わせることで、高い変異効率を達成しつつ、非特異的な変異誘発を抑制することができることが示された。
複数のコピー因子は、相当量のゲノム配列を占める。組換え、又はゲノム切断を含む他の方法とは異なり、Target-AIDはゲノムの不安定性を誘導することなく、同じsgRNA配列を用いて複数の遺伝子座を一度に編集し得る。このコンセプトを証明するため、大腸菌ゲノム中の4個の主要な転移因子(TE:IS1,2,3及び5)を、4個のsgRNAを用いて同時に標的化した。IS1,2,3及び5のための、それぞれ10個、12個、5個及び14個の遺伝子座は、各遺伝子座についてユニークなPCRプライマーにより特異的に増幅することができた。sgRNAは、各TEのトランスポザーゼ遺伝子の共通配列を含むように設計し、終止コドンを導入した(図11)。大腸菌Top10細胞を、dCas-CDA-UGI-LVA及び4個の標的sgRNAをそれぞれ発現する2個のプラスミドで形質転換した。ISを編集した細胞の分離と検証の手順を図12に示し、以下に詳細に説明する。二重形質転換及び選択の後、コロニーをPCRで増幅し、まずIS5-1、IS5-2、IS5-11、IS5-12の部位でシーケンシングした。IS5標的は効率が悪いことが判明した。分析された4つのコロニーのうち、1つは3箇所の変異部位及び1つの異種遺伝子型の部位を含んでいた(IS5-1)。次いで、細胞を液体培地に懸濁し、プレート上に広げてコロニーを再度単離した。8個のコロニーのうち3個はIS5-1に変異を含み、そのうちの2個について、残り24個のIS遺伝子座の配列をさらにシーケンシングしたところ、全ての変異した部位を含むが、1つの不完全な、異種遺伝子型の部位(IS5-5)を含むことが示された。次いで、細胞を懸濁させて広げ、IS5-5に変異を含む6個の再単離したクローンのうちの4個のクローンを得た。そのクローンの1個をIS5部位でシーケンシングしたところ、1個の異種遺伝子型の部位(IS5-2)を含むことが判明した。8個のクローンを再度単離し、6個のクローンがIS5-2での変異を含んでいた。2個のクローンを非選択培地上に広げて、プラスミドを失った細胞を得た。次いで、細胞をゲノム抽出して、シーケンシングし、全てのIS部位で変異を確認し(図11)、さらにゲノム全体のオフターゲット効果を評価するために、全ゲノムシーケンシングを行った。PAMに近接した1~8塩基で一致した配列を含む参照ゲノムからの34箇所の潜在的なオフターゲット部位のうち、2箇所の部位が変異していることが判明した(表5)。
Casエフェクタータンパク質としてLbCpf1(配列番号326及び327)を、タンパク質分解タグとしてYAASV及びYALAAをコードする酵母発現用ベクター(ベクター3685:Cpf1-NLS-3xFlag-YAASV(配列番号328)、ベクター3687:Cpf1-NLS-3xFlag-YALAA(配列番号329))、及びタンパク質分解タグをコードする核酸を有さないコントロールベクター(ベクター3687:Cpf1-NLS-3xFlag(配列番号330))を、pRS315ベクターをベースとして作成した。これらのベクターを用いて、大腸菌の形質転換効率を検証した。図13に、各ベクターの概略図を示す。下記表6に示す通り、各ベクターを含むDNA溶液を2ng/μlに調整し、20μlの大腸菌Top10コンピテントセルに、1μl(2ng)のDNA溶液を加えて形質転換した。その後、200μlのSOCを加えて37℃1時間回復培養し、氷上で5分おいて増殖停止した後に、1μlの 50mg/ml Ampを加えた。培養液の一部(1μl及び10μl)をTEで希釈してLB+Ampプレートに塗布し、37℃で一晩培養してコロニー数を計測した。結果を表6に示す。
Claims (20)
- 二本鎖DNA中の標的ヌクレオチド配列と特異的に結合する核酸配列認識モジュールと、(i)疎水性アミノ酸3残基をC末端に含むペプチド、又は(ii)該アミノ酸残基の少なくとも一部がセリンに置換されたアミノ酸3残基をC末端に含むペプチドからなるタンパク質分解タグとが結合した、複合体。
- 前記複合体が、核酸改変酵素がさらに結合した複合体であって、標的化された部位の1以上のヌクレオチドを他の1以上のヌクレオチドに変換し又は欠失させ、あるいは該標的化された部位に1以上のヌクレオチドを挿入する、請求項1に記載の複合体。
- 前記アミノ酸3残基が、ロイシン-バリン-アラニン、ロイシン-アラニン-アラニン、アラニン-アラニン-バリン又はアラニン-セリン-バリンである、請求項1又は2に記載の複合体。
- 前記核酸配列認識モジュールが、Casの2つのDNA切断能のうちの一方のみ、又は両方のDNA切断能が失活したCRISPR-Casシステムである、請求項1~3のいずれか1項に記載の複合体。
- 前記複合体が、CRISPR-Casシステムと、タンパク質分解タグとが結合した複合体である、請求項1~3のいずれか1項に記載の複合体。
- 前記核酸改変酵素が核酸塩基変換酵素又はDNAグリコシラーゼである、請求項2~4のいずれか1項に記載の複合体。
- 前記核酸塩基変換酵素がデアミナーゼである、請求項6に記載の複合体。
- 塩基除去修復のインヒビターがさらに結合した、請求項6又は7に記載の複合体。
- 請求項1~8のいずれか1項に記載の複合体をコードする核酸。
- 細菌の二本鎖DNAの標的化された部位を改変する、又は該部位の近傍で二本鎖DNAにコードされる遺伝子の発現を調節する方法であって、選択された二本鎖DNA中の標的ヌクレオチド配列と特異的に結合する核酸配列認識モジュールと、(i)疎水性アミノ酸3残基をC末端に含むペプチド、又は(ii)該アミノ酸残基の少なくとも一部がセリンに置換されたアミノ酸3残基をC末端に含むペプチドからなるタンパク質分解タグとが結合した複合体を、該二本鎖DNAと接触させる工程を含む、方法。
- 前記複合体が、核酸改変酵素がさらに結合した複合体であって、該標的化された部位の1以上のヌクレオチドを他の1以上のヌクレオチドに変換する又は欠失させる、あるいは該標的化された部位に1以上のヌクレオチドを挿入する工程を含む、請求項10に記載の方法。
- 前記アミノ酸3残基が、ロイシン-バリン-アラニン、ロイシン-アラニン-アラニン、アラニン-アラニン-バリン又はアラニン-セリン-バリンである、請求項10又は11に記載の方法。
- 前記核酸配列認識モジュールが、Casの2つのDNA切断能のうちの一方のみ、又は両方のDNA切断能が失活したCRISPR-Casシステムである、請求項10~12のいずれか1項に記載の方法。
- 前記複合体が、CRISPR-Casシステムと、タンパク質分解タグとが結合した複合体である、請求項10~12のいずれか1項に記載の方法。
- 異なる標的ヌクレオチド配列とそれぞれ特異的に結合する、2種以上の核酸配列認識モジュールを用いることを特徴とする、請求項10~14のいずれか1項に記載の方法。
- 前記異なる標的ヌクレオチド配列が、異なる遺伝子内に存在する、請求項15に記載の方法。
- 前記核酸改変酵素が核酸塩基変換酵素又はDNAグリコシラーゼである、請求項10~13、15及び16のいずれか1項に記載の方法。
- 前記核酸塩基変換酵素がデアミナーゼである、請求項17に記載の方法。
- 該複合体が、塩基除去修復のインヒビターがさらに結合したものである、請求項17又は18に記載の方法。
- 二本鎖DNAと複合体との接触が、該二本鎖DNAを有する細菌への、該複合体をコードする核酸の導入により行われる、請求項10~19のいずれか1項に記載の方法。
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JPWO2019103020A1 (ja) | 2021-01-21 |
CA3082922A1 (en) | 2019-05-31 |
JP7328695B2 (ja) | 2023-08-17 |
KR102387830B1 (ko) | 2022-04-18 |
JP2023129709A (ja) | 2023-09-14 |
KR20200091884A (ko) | 2020-07-31 |
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