WO2022210748A1 - Nouveau polypeptide ayant la capacité de former un complexe avec un arn guide - Google Patents

Nouveau polypeptide ayant la capacité de former un complexe avec un arn guide Download PDF

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WO2022210748A1
WO2022210748A1 PCT/JP2022/015608 JP2022015608W WO2022210748A1 WO 2022210748 A1 WO2022210748 A1 WO 2022210748A1 JP 2022015608 W JP2022015608 W JP 2022015608W WO 2022210748 A1 WO2022210748 A1 WO 2022210748A1
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polypeptide
present
guide rna
amino acid
seq
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良純 石野
園子 石野
裕之 松本
隆司 上森
武宏 相良
健介 安部
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国立大学法人九州大学
タカラバイオ株式会社
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Publication of WO2022210748A1 publication Critical patent/WO2022210748A1/fr

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Definitions

  • the present invention relates to a polypeptide capable of forming a complex with a guide RNA, particularly a thermostable Cas nuclease (CRISPR-related enzyme).
  • CRISPR-related enzyme thermostable Cas nuclease
  • CRISPR clustered regularly interspaced short palindromic repeat
  • CRISPR-associated (cas) gene clusters encoding nucleases and helicases are present near the CRISPR repeats (Non-Patent Document 1).
  • CRISPR loci there are three elements: the cas gene group, the preceding sequence, and the repeat/spacer sequence.
  • it is roughly classified into two classes according to the type of Cas protein group that constitutes the CRISPR-Cas system and the difference in action mechanism, and each CRISPR-Cas system is classified into several subtypes.
  • Class 1 is subdivided into Types I, III, and IV
  • Class 2 is subdivided into Types II, V, and VI, respectively.
  • Cas12a is classified as Class 2, Type V CRISPR-Cas (Non-Patent Document 2).
  • Non-Patent Document 3 A nuclease Cpf1 was discovered in the bacterium Francisella novicida (Non-Patent Document 3, Patent Document 1). Originally named Cpf1, it was renamed Cas12a in line with the CRISPR-Cas system classification.
  • Non-Patent Document 5 In addition to genome editing, the CRISPR-Cas9 system is being considered for various applications such as transcription control, imaging, and nucleic acid detection.
  • An object of the present invention is to provide a novel thermostable Cas nuclease.
  • thermostable Cas nuclease surprisingly found that a nucleic acid encoding a thermostable Cas nuclease exists in a microbial group collected from hot spring samples in Oita Prefecture. Found it. Thus, the present invention was completed.
  • the present invention [1] the amino acid sequence of SEQ ID NO: 1, An amino acid sequence having 1 to 10 amino acid mutations in the amino acid sequence of SEQ ID NO: 1, wherein said mutations are substitutions, deletions, insertions and/or additions, and 50% or more identity with the sequence of SEQ ID NO: 1 an amino acid sequence having an isolated polypeptide capable of complexing with a guide RNA comprising a sequence selected from the group consisting of; [2] The polypeptide of [1], which further has nuclease activity; [3] The polypeptide according to [1] or [2], which is a polypeptide classified as class 2 type V CRISPR/Cas; [4] The polypeptide of any one of [1] to [3], which has the activity of cleaving double-stranded DNA and single-stranded DNA at 55-65°C; [5] contains an amino acid sequence in which amino acid substitutions, deletions, insertions, and/or additions have occurred in the amino acid sequence of the poly
  • a novel thermostable Cas nuclease and a method for producing the same are provided by the present invention.
  • a Cas nuclease Cas12a belonging to Class 2, Type V (hereinafter referred to as the polypeptide of the present invention) is provided.
  • Such heat-resistant Cas12a was not known, and was discovered for the first time by the present invention.
  • 1 shows the results of target double-stranded DNA cleavage activity measurement using the polypeptide of the present invention.
  • the results of the dissociation measurement of the complex (RNP) and target double-stranded DNA are shown.
  • 1 shows the results of heat resistance measurement of the polypeptide of the present invention.
  • 1 shows the results of optimal temperature measurement of the polypeptide of the present invention.
  • 1 shows the results of measuring the length of the target double-stranded DNA of the polypeptide of the present invention.
  • 1 shows the results of measurement of single-strand DNA cleavage activity of the polypeptide of the present invention.
  • 4 shows the analysis results of the PAM sequences recognized by the polypeptides of the present invention.
  • 1 shows the results of measuring target double-stranded DNA cleavage activity using the polypeptide lacking nuclease activity of the present invention. 1 shows the results of target single-strand DNA cleavage activity measurement using the polypeptide lacking nuclease activity of the present invention. 1 shows the results of specific detection of target genes using the polypeptides of the present invention.
  • Polypeptides of the Present Invention (1) Polypeptides of the Present Invention A first aspect of the present invention relates to polypeptides capable of forming a complex with a guide RNA.
  • a protein comprising the amino acid sequence of SEQ ID NO: 1 is exemplified as the polypeptide of the present invention.
  • the amino acid sequence was classified as Cas12a from the cluster structure and the like, but as a result of BLAST search, it was a novel amino acid sequence with only low identity with known Cas nucleases. In addition, there was no region with particularly high identity.
  • the polypeptide with the highest homology was Cpf1 (GenBank: PJE64038.1), with about 26% identity.
  • polypeptide of the present invention SEQ ID NO: 1 and at least 50% or more, for example, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more , 95% or more, 98% or more, or 99% or more, and having the ability to form a complex with a guide RNA are also included in the polypeptides of the present invention.
  • 1 to 10 for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in SEQ ID NO: 1 are substituted, deleted, inserted, and/or added
  • the polypeptides of the present invention also include polypeptides having the same amino acid sequence and the ability to form a complex with a guide RNA.
  • the polypeptide of the present invention is not particularly limited to the presence or absence of nuclease activity as long as it has the ability to form a complex with guide RNA.
  • heat resistance refers to the property of being able to have enzymatic activity even after heat treatment. For example, retains 50% or more activity, such as the ability to form a complex with guide RNA, compared to before treatment even after treatment for 5 minutes at a temperature of 55°C or higher, 65°C or higher, or 75°C or higher. It can be said that it "has heat resistance” when it is possible to do so.
  • the polypeptide of the present invention exhibits activity even at temperatures of 40°C or higher, for example, 45°C or higher, 55°C or higher, 65°C or higher, or 75°C or higher. Moreover, the polypeptide of the present invention exhibits the highest activity at a high temperature range of 55-65°C. Even after being kept warm in the high temperature range, it has the same activity as before the reaction.
  • the polypeptide of the present invention has an activity of forming a complex with a guide RNA to cleave a specific portion of a target double-stranded DNA, and forming a complex with a guide RNA and not being a target nucleic acid. It has the activity of non-specifically cleaving single-stranded DNA at any site. Cleavage of the target double-stranded DNA occurs through the action of the polypeptide of the present invention having nuclease activity and guide RNA. On the other hand, cleavage of single-stranded DNA occurs in the polypeptide of the present invention alone, following cleavage of the target double-stranded DNA.
  • the complex of the polypeptide of the present invention and guide RNA (hereinafter sometimes referred to as RNP) is very stable compared to known Cas9-guide RNA complexes. Whereas the known complexes tend to dissociate, the complexes of the polypeptide and guide RNA of the present invention are 40°C or higher, for example, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, or 70°C or higher. The complex remains formed even under temperature conditions of °C or higher.
  • polypeptide of the present invention and the guide RNA may be covalently bound.
  • the method of covalent bonding there is no limitation on the method of covalent bonding, and any method commonly used in this field can be used.
  • covalently binding it may be via a linker.
  • a polypeptide of the invention and a guide RNA may be irreversibly crosslinked.
  • polypeptide of the present invention may lack nuclease activity.
  • Such polypeptides utilize the specific base sequence recognition ability of complexes with guide RNA, and are used, for example, for separation and purification of nucleic acid fragments having specific base sequences by using immunoprecipitation technology together. can do.
  • Guide RNA Guide RNA refers to RNA that guides the Cas nuclease protein to the target DNA sequence in the CRISPR-Cas system, and is the nucleic acid that determines the DNA sequence specificity of the CRISPR-Cas system.
  • the guide RNA and Cas nuclease form a complex, recognize the PAM (protospacer adjacent motif) sequence and bind to the target DNA, further anneal the guide RNA to the target DNA sequence, and the Cas nuclease cleaves the target DNA sequence.
  • PAM protospacer adjacent motif
  • the guide RNA to which Cas12a binds consists only of crRNA (CRISPR RNA) having a sequence complementary to the target DNA sequence to be cleaved.
  • the guide RNA for other Cas nucleases such as Cas9 consists of two types of RNA molecules: crRNA and tracrRNA (trans-activating crRNA) that serves as a base for forming a complex with Cas nuclease.
  • sgRNA single guide RNA
  • Casl2a differs from other Cas nucleases such as Cas9.
  • the guide RNA sequence is preferably designed to be the base sequence on the 5' (upstream) side or 3' (downstream) side of the PAM sequence present in the target DNA sequence to be cleaved.
  • the design of the guide RNA sequence does not limit the present invention, for example, known online tools (CHOPChop (https://chopchop.cbu.uib.no/), CRISPRdirect (https://crispr.dbcls. jp/), CRISPR Design Tool (https://horizondiscovery.com/products/tools/CRISPR-Design-Tool/), etc.) can be suitably used.
  • the guide RNA sequence may be any sequence that can anneal with the target DNA sequence while forming a complex with the polypeptide of the present invention. Also, any sequence may be used as long as it can cleave the target DNA sequence in the state of forming a complex with the polypeptide of the present invention having nuclease activity.
  • the length of the guide RNA does not limit the present invention, it is preferably 17mer or longer, 20mer or longer, or 22mer or longer. In addition, although the length of the guide RNA does not limit the present invention, it is preferably 100 mer or less, 80 mer or less, or 60 mer or less.
  • PAM sequence PAM sequence is a sequence for Cas nuclease to recognize a target DNA sequence.
  • the PAM sequence differs depending on the type of Cas nuclease and the biological species from which it is derived.
  • Acidaminococcus sp. Cpf1 derived hereinafter sometimes abbreviated as AsCpf1
  • Cpf1 derived from Lachnospiraceae bacterium hereinafter sometimes abbreviated as LbCpf1
  • AsCpf1 derived or Cpf1 derived from Lachnospiraceae bacterium
  • LbCpf1 is 5′-TTTV-3′, 5′-TTCA-3′, 5′-TCTA- Recognizes T-rich sequences such as 3', 5'-CTTA-3' (T.
  • Cpf1 cannot recognize a sequence containing two or more Cs as a PAM sequence.
  • Cas9 recognizes G-rich sequences such as 5'-NGG-3' and 5'-NGGRR(T)-3'.
  • V means not T (A, C, or G)
  • N means (A, C, G, or T)
  • R means (A or G).
  • the PAM sequence of the polypeptide of the present invention can be determined by a known method, for example, the method described in Examples below.
  • the PAM sequence of the polypeptide of the invention having the amino acid sequence of SEQ ID NO: 1 is YYN.
  • AsCpf1 and LbCpf1 which are also classified into Cas12a
  • the polypeptide of the present invention can also recognize a sequence containing two Cs as a PAM sequence.
  • Y is (C or T).
  • the polypeptide of the present invention and the guide RNA When acting on the target DNA, the polypeptide of the present invention and the guide RNA may be prepared as separate molecules so that they form a complex when they are mixed, or they may be mixed in advance to form a complex.
  • the body may be formed.
  • the polypeptide of the invention and guide RNA may be covalently bound and used as one molecule. Any binding mode between the polypeptide of the present invention and the guide RNA is acceptable as long as it can bind to a target DNA sequence having a sequence complementary to the guide RNA.
  • a molecule obtained by covalently binding the polypeptide of the present invention and a guide RNA is also included in the complex of the present invention.
  • a second aspect of the invention relates to nucleic acids encoding the polypeptides of the invention. Specifically, nucleic acids encoding the polypeptides of the present invention.
  • a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 is exemplified as a nucleic acid encoding the polypeptide of the present invention. Also, an example thereof is a nucleic acid containing the base sequence of SEQ ID NO:2.
  • the nucleotide sequence of the nucleic acid encoding the polypeptide of the present invention is not particularly limited as long as it encodes the polypeptide of the present invention. may be included.
  • a nucleic acid designed by selecting codons (optimizing codons) according to the host to be used may be used as appropriate. .
  • the codon optimization can be performed by a method commonly used in this field.
  • a third aspect of the invention relates to an expression vector comprising a nucleic acid encoding a polypeptide of the invention.
  • the expression vector of the present invention preferably contains a nucleic acid encoding the polypeptide of the present invention and an expression control sequence operably linked to the nucleic acid.
  • the expression vector carrying the nucleic acid encoding the polypeptide of the present invention is not particularly limited as long as it is an expression vector commonly used in this field.
  • a vector capable of autonomous replication in a host cell or a vector that can be integrated into the host chromosome can be used. That is, an expression vector compatible with the host may be used.
  • an expression vector carrying a nucleic acid encoding the polypeptide of the present invention for example, a plasmid vector, a phage vector, a virus vector, etc. can be used. These vectors are well known to those skilled in the art, and many are commercially available. These known vectors and variants thereof can be used in the present invention.
  • the promoter to be loaded into the expression vector of the present invention can be selected according to the host. can be used, but are not limited to those listed above. Furthermore, an expression system (eg, pET expression system, etc.) combining a phage-derived promoter and an RNA polymerase gene may be used.
  • an expression system eg, pET expression system, etc.
  • the expression vector of the present invention may further contain a nucleic acid encoding an affinity tag.
  • a nucleic acid encoding an affinity tag is inserted into a vector so that a fusion protein of the polypeptide of the present invention and the affinity tag is expressed.
  • the position at which the nucleic acid encoding the tag is added may be either the 5'-end and/or the 3'-end of the nucleic acid encoding the polypeptide of the present invention, and the expression of the fusion protein and the function of the polypeptide of the present invention are enhanced. It may be appropriately added to a position that does not impede the holding and tagging functions.
  • the tag may be a tag that can be removed during or after purification of the expressed polypeptide.
  • the expression vector of the present invention may contain one or more expression control sequences in addition to the promoter.
  • the expression regulatory sequences are not particularly limited, but include genes involved in promoter regulation, ribosome binding sequences, polyadenylation signals, transcription termination sequences (transcription terminators), enhancers and the like.
  • the expression vector of the present invention contains a gene encoding a replication origin and a marker (drug resistance gene, fluorescent marker, luminescent marker, etc.) used for selection of transformants, and a base sequence for enhancing translation efficiency. may contain.
  • the fourth aspect of the present invention relates to cells transformed with the above expression vector.
  • the cell (host) transformed with the vector expressing the polypeptide of the present invention is not particularly limited as long as it is a host commonly used in this field.
  • bacteria Esscherichia coli, Bacillus subtilis, etc.
  • yeast filamentous fungi
  • insect cells eukaryotic cells
  • animal cells mammalian cells including human cells, etc.
  • These hosts are well known to those skilled in the art, and many are commercially available. These known hosts and variants thereof can be used in the present invention.
  • the method for introducing the expression vector into the host is not particularly limited as long as it is a method capable of introducing the expression vector into the host. Examples include methods using calcium ions, electroporation, spheroplast method, and lithium acetate. method, calcium phosphate method, lipofection method and the like can be used. These introduction methods are well known to those skilled in the art, and equipment and kits for them are commercially available.
  • a transformant obtained by introducing an expression vector into a host is cultured, and the polypeptide of the present invention can be obtained from the culture.
  • Culture conditions are not particularly limited as long as they are suitable for the expression vector, host, etc. used.
  • a necessary inducer may be added at an appropriate timing depending on the type of host or expression vector used.
  • the culture solution is centrifuged, and the obtained cells are washed and subjected to ultrasonic disruption, freeze-thaw treatment, bacteriolytic enzyme treatment, or the like to obtain a fragment containing the polypeptide of the present invention.
  • the present invention can be purified by appropriately combining purification methods used in this field, such as ammonium sulfate precipitation, anion exchange column, cation exchange column, gel filtration column, affinity chromatography column, dialysis, etc. It may be desirable to purify the polypeptides of the invention. These culture methods and purification methods are well known to those skilled in the art, and equipment and kits therefor are commercially available. A method for producing the polypeptide of the present invention using the transformant is also an aspect of the present invention.
  • compositions or Kits of the Invention A fifth aspect of the invention relates to compositions or kits comprising a polypeptide of the invention.
  • composition of the present invention is a composition for cleaving nucleic acids.
  • compositions include guide RNA, buffer components, sterile water, etc., in addition to the polypeptides of the present invention having nuclease activity.
  • other components necessary for nucleic acid cleavage reactions such as divalent metal salts, may be further contained.
  • kits of the present invention are kits for cleaving nucleic acids.
  • the kit of the present invention is, for example, a kit containing the composition of the present invention. That is, the kit of the present invention contains, in addition to the polypeptide of the present invention having nuclease activity, components necessary for nucleic acid cleavage reaction, such as guide RNA, divalent metal salts, buffer components, sterilized water, and the like.
  • another embodiment includes a kit for purifying, separating, and detecting a nucleic acid fragment having a specific base sequence. It contains a solid-phase carrier bound with an antibody capable of specifically recognizing the polypeptide, a buffer component, sterilized water, and the like.
  • composition or kit of the present invention may contain components necessary for a PCR reaction system, such as a PCR primer set for amplifying the target nucleic acid, reverse transcriptase, thermostable DNA polymerase, inorganic pyrophosphatase, and the like. good.
  • a component necessary for detection such as a labeled reporter nucleic acid.
  • the sixth aspect of the present invention relates to a method for cleaving a target nucleic acid using the polypeptide of the present invention having nuclease activity.
  • polypeptide of the present invention having nuclease activity can be used together with a guide RNA to specifically cleave a specific portion of a target double-stranded DNA.
  • a polypeptide of the invention may be provided in the form of a complex with a guide RNA, or the polypeptide of the invention and guide RNA may be provided as separate molecules and allowed to form a complex in situ. good too.
  • the polypeptide of the present invention has heat resistance that exhibits activity even under temperature conditions of 40° C. or higher, for example, 45° C. or higher, 50° C. or higher, 55° C. or higher, 65° C. or higher, or 75° C. or higher.
  • Reactions can be performed under higher temperature conditions compared to known Cas nucleases. Therefore, it can be used in reaction systems that could not be used due to the inactivation of known Cas nucleases.
  • the reaction can be carried out at a high temperature, the secondary structure of the nucleic acid can be dissociated, and the efficiency of cleaving the target nucleic acid can be improved.
  • the complex of the polypeptide of the present invention and guide RNA can non-specifically cleave single-stranded DNA in addition to the above-described cleavage of double-stranded DNA. Therefore, the polypeptide of the present invention, when used with a guide RNA, non-specifically cleaves single-stranded DNA in addition to cleaving double-stranded DNA. Therefore, the complex of the polypeptide of the present invention and guide RNA can be used, for example, in a detection system using a probe having a fluorescent dye and a quenching group, coexisting with the target double-stranded DNA.
  • the seventh aspect of the invention relates to the introduction of the polypeptides of the invention into target cells.
  • the complex of the polypeptide of the present invention and guide RNA is very stable. As a result, when the complex is introduced into target cells by electroporation, the introduction procedure can be repeated to maintain high efficiency of introduction of the complex into cells. In addition, even when other introduction techniques are used, introduction of the complex into cells can be achieved with high efficiency.
  • the complex can be incorporated into the cells as it is formed. It is possible. That is, a step of strict washing of the target cells becomes unnecessary. Since the man-hours can be reduced and the reagents used can be reduced, contamination can be prevented, and it is possible to maintain a high state of cell proliferation and viability.
  • a vector containing a nucleic acid encoding the polypeptide of the present invention and a nucleic acid encoding a guide RNA may be used for introduction into target cells.
  • the vector does not limit the present invention in any way, for example, a plasmid vector or a virus vector can be preferably used.
  • the eighth aspect of the present invention relates to genome editing.
  • Genome editing of target cells is possible by introducing a complex of the polypeptide of the present invention having nuclease activity and guide RNA into target cells. All of base substitution, knock-in, and knock-out are possible as genome editing.
  • a method of introducing a nucleic acid encoding the polypeptide of the present invention and a guide RNA into a cell to express both in the cell, a nucleic acid encoding the polypeptide of the present invention. and a guide RNA into a cell, or a method of introducing a polypeptide of the present invention and a nucleic acid encoding the guide RNA into a cell.
  • nucleic acid constructs are designed so that they are expressed in the cell.
  • the ninth aspect of the present invention relates to use for target nucleic acid detection.
  • the polypeptides of the present invention can be used with guide RNA to detect target nucleic acids.
  • a polypeptide of the invention may have nuclease activity or may lack nuclease activity.
  • the known Cas nuclease is thermolabile, and in order to incorporate it into the target nucleic acid detection system, it is added after the target nucleic acid is amplified. That is, the lid of the container is opened during the detection process, and there is a risk of contamination.
  • the polypeptide of the present invention is heat-resistant, and for example, when a product amplified by PCR is used as the target nucleic acid, it can be added in the step of mixing reagents for PCR first. This eliminates the risk of contamination mentioned above.
  • the reaction can be carried out at a high temperature, the secondary structure of the nucleic acid can be dissociated, and the efficiency of cleaving the target nucleic acid can be improved.
  • a composition for detecting a target nucleic acid includes, in addition to the polypeptide of the present invention, a guide RNA, a buffer component, sterilized water, a PCR primer set for amplifying the target nucleic acid, a reverse transcriptase, a DNA polymerase, and a labeled reporter. It includes nucleic acids, inorganic pyrophosphatase, and the like. When the polypeptide of the present invention further has nuclease activity, it may further contain components necessary for nucleic acid cleavage reaction, such as divalent metal salts.
  • a tenth aspect of the present invention relates to use for NGS library preparation.
  • polypeptides of the present invention can be used for degrading and removing non-analytical nucleic acids, and for concentrating and recovering target nucleic acids in the preparation of libraries for use in next-generation sequencing (NGS).
  • NGS next-generation sequencing
  • polypeptides of the present invention are useful in reducing this noise-causing nucleic acid.
  • polypeptides of the Invention Deficient in Nuclease Activity
  • An eleventh aspect of the invention relates to polypeptides of the invention deficient in nuclease activity.
  • Mutants of the polypeptide of the present invention lacking nuclease activity (hereinafter sometimes referred to as dead Cas) lack nuclease activity but retain the ability to bind to target double-stranded DNA. It remains the same and is a variant that can be used as a protein domain that specifically recognizes DNA sequences.
  • the aforementioned mutants can be produced by introducing mutations into the nuclease domains in the polypeptides of the present invention by amino acid substitutions to eliminate the nuclease activity.
  • the RuvC-I domain is at positions 920-977
  • the RuvC-II domain is at positions 996-1105
  • the RuvC-III domain is at positions 1305-1353.
  • the mutation is a substitution of one or more amino acid residues (eg, 1-10 amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) , deletions, insertions or additions, or combinations thereof, as long as they lack nuclease activity.
  • amino acid residues eg, 1-10 amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues
  • the dead Cas can be expected to be used differently from the wild type due to the lack of nuclease activity.
  • the present invention is not limited in any way, it can be used for the following applications, for example.
  • CRISPRi steric hindrance
  • a fusion of dead Cas and a transcriptional activation factor is bound to the promoter region to activate transcription (CRISPRa; CRISPR activation).
  • CRISPRa CRISPR activation
  • Using dead Cas in combination with a demethylating agent low-molecular-weight compound, enzyme, etc.
  • the dead Cas of the present invention is such that the amino acid sequence of a polypeptide capable of forming a complex with a guide RNA, selected from the following (a) to (c), lacks the nuclease activity of the polypeptide.
  • a polypeptide containing a mutated amino acid sequence is meant. (a) the amino acid sequence of SEQ ID NO: 1; (b) an amino acid sequence having 1 to 10 amino acid mutations in the amino acid sequence of SEQ ID NO: 1, wherein said mutations are substitutions, deletions, insertions and/or additions; and (c) the sequence of SEQ ID NO: 1 and Amino acid sequences with 50% or more identity.
  • the dead Cas of the present invention includes, for example, an amino acid sequence containing a substitution of aspartic acid to alanine for the 945th amino acid residue in the amino acid sequence of SEQ ID NO: 1, and a guide RNA
  • a polypeptide that has the ability to form a complex with and lacks nuclease activity is exemplified.
  • Such polypeptides include, for example, a polypeptide comprising the amino acid sequence shown in SEQ ID NO:6.
  • amino acid sequence of SEQ ID NO: 6 in which the 945th aspartic acid in the amino acid sequence of SEQ ID NO: 1 is replaced with alanine, substitution, deletion, or insertion of (preferably up to 10) other amino acid residues
  • substitution, deletion, or insertion of (preferably up to 10) other amino acid residues A polypeptide having an amino acid sequence containing and/or additions and having the ability to form a complex with a guide RNA is also included in the dead Cas of the present invention.
  • glutamic acid at position 1030, arginine at position 1255, or aspartic acid at position 1305 in the amino acid sequence of SEQ ID NO: 1 also has the ability to form a complex with guide RNA by substituting alanine for the amino acid residue. and lacking nuclease activity can be produced. Moreover, if necessary, a plurality of the above amino acid substitutions may be combined.
  • a nucleic acid encoding the dead Cas polypeptide of the present invention is a nucleic acid having a nucleotide sequence encoding the above polypeptide, and one embodiment thereof is a nucleic acid encoding the amino acid sequence of SEQ ID NO:6. Also, an example thereof is a nucleic acid containing the nucleotide sequence of SEQ ID NO:7. As long as it encodes the dead Cas polypeptide of the present invention, there is no limitation on the nucleotide sequence of the nucleic acid encoding the dead Cas polypeptide of the present invention. It may contain a codon different from number 7.
  • the dead Cas polypeptide of the present invention using the nucleic acid of the present invention, using a nucleic acid designed by appropriately selecting codons (codon optimization) according to the host to be used Also good.
  • the codon optimization can be performed by a method commonly used in this field.
  • Example 1 Metagenome analysis of hot spring water (1) Metagenome analysis was performed on the microbial groups contained in the hot water samples from Kamado Jigoku in Beppu City, Oita Prefecture. First, a DNA specimen extracted from a sample was physically fragmented into several hundred bp using an acoustic stabilizer Covaris (Covaris) and size-selected using AMPure (registered trademark) XP (Beckman Coulter). A library was prepared from this fragment using Thru PLEX (registered trademark) DNA-Seq Kit (Takara Bio USA) and used for sequence analysis.
  • Covaris acoustic stabilizer Covaris
  • AMPure registered trademark
  • XP Bacillus Coulter
  • MiSeq (illumina Inc.) was used for sequence analysis, and paired-end analysis of 250 bp length was used as a condition. After assembly of the sequence analysis results, a novel gene (SEQ ID NO: 2) was found in the contig in the library. The novel gene encoded a polypeptide having the amino acid sequence of SEQ ID NO:1.
  • homologues of cas4, cas1, and cas2 genes are arranged in the downstream region of the novel gene of SEQ ID NO: 2, the novel gene of SEQ ID NO: 2 is judged to be a CRISPR-associated protein belonging to class 2, type V. did.
  • homologues of CRISPR1, CRISPR2, CRISPR3, and CRISPR4 genes were arranged in the further downstream region.
  • Example 2 Expression of Polypeptide of the Present Invention
  • (1) Construction of Expression Plasmid In order to analyze the function of the polypeptide having the amino acid sequence of SEQ ID NO: 1, a forced expression system in Escherichia coli was constructed.
  • the artificially synthesized nucleic acid of SEQ ID NO: 2 was inserted into the in-fusion cloning site of the expression vector pET6xHN-N (Takara Bio USA) according to the instruction manual attached to the kit.
  • Escherichia coli BL21 DE3 strain was transformed with the resulting plasmid, and ampicillin-resistant clones were selected. From this clone was obtained the plasmid pET-Cas12a-N for expression of the polypeptides of the invention.
  • the artificially synthesized nucleic acid of SEQ ID NO: 2 was inserted into the in-fusion cloning site of the expression vector pET6xHN-C (Takara Bio USA).
  • pET6xHN-C Takara Bio USA.
  • the expression vector pET6xHN-N has a His tag attached to the N-terminal side of the expressed protein
  • pET6xHN-C has a structure in which a His tag is attached to the C-terminal side of the expressed protein.
  • the obtained suspension after sonication was heated at 95°C for 10 minutes to denature the heat-labile protein. Then, it was centrifuged at 9500 xg for 30 minutes at 4°C, and the supernatant was recovered. Imidazole was added to the supernatant to a final concentration of 50 mM (hereinafter referred to as crude extract).
  • a Ni Sepharose FF column was loaded with 3 times the amount of sterilized water, 3 times the amount of 200 mM NiCl 2 , 3 times the amount of sterilized water, and 3 times the amount of Buffer A (50 mM Tris-HCl pH 7.5, 500 mM NaCl , 5% glycerol, and 50 mM imidazole) were applied sequentially to equilibrate the column.
  • Buffer A 50 mM Tris-HCl pH 7.5, 500 mM NaCl , 5% glycerol, and 50 mM imidazole
  • the resulting crude extract was applied to an equilibrated column.
  • the column was washed with 4 times the amount of Buffer A as the crude extract. Thereafter, concentration gradient elution was performed using Buffer A containing 50 mM to 300 mM imidazole to elute the adsorbate from the column.
  • Buffer B 50 mM Tris-HCl pH 7.5, 500 mM NaCl, and 5% glycerol
  • a 20 mM KPB (dipotassium hydrogen phosphate and potassium dihydrogen phosphate)/buffer B mixture of 10 times the amount of resin was applied to a hydroxyapatite column (CHT Ceramic Hydroxypatite Type I 80 um) and equilibrated.
  • the obtained dialysate was applied to a hydroxyapatite column.
  • the column was washed with 20 mM KPB/Buffer B, 5 times the amount of the dialysate. Thereafter, concentration gradient elution was performed using Buffer B containing 20 mM to 300 mM KPB to elute the adsorbate from the hydroxyapatite column.
  • the purified polypeptide of the present invention was not contaminated with host-derived nucleases. Measurements were performed for RNase activity (substrates are 16S rRNA and 23S rRNA), DNase activity (substrate is ⁇ -DNA), endonuclease activity (substrate is pBR322 plasmid), and exonuclease activity (substrate is ⁇ -HindIII digest). As a result, no activity was detected.
  • the composition of the reaction solution used was 50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, and 0.1 mg/ml BSA (all final concentrations).
  • each subsequent test used the purified polypeptide of the present invention.
  • the expressed protein with a His tag attached to the N-terminal side was referred to as Cas12a-N
  • the expressed protein with a His tag attached to the C-terminal side was referred to as Cas12a-C.
  • Example 3 Activity measurement 18-1000 ng of the above purified polypeptide (Cas12a-N or Cas12a-C) and 2 pmol of guide RNA (SEQ ID NO: 3) were mixed and heated at 37°C for 5 minutes. Next, 100 ng of the substrate double-stranded DNA (SEQ ID NO: 4) was mixed and reacted in the following order. (1) heating at 37° C. for 1 hour, (2) heating at 80° C. for 5 minutes, and (3) keeping the temperature at 4° C. after the reaction. The composition of the reaction solution used was 50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, and 0.1 mg/ml BSA (all final concentrations).
  • the liquid volume after mixing is 20 ⁇ l.
  • the same composition and volume of the reaction solution were used.
  • a portion of the reaction solution was then subjected to agarose gel electrophoresis to examine the presence or absence of cleavage of the substrate DNA.
  • the substrate double-stranded DNA is part of the human DNMT1 gene (GeneID: 1786). Specifically, it is a base sequence corresponding to positions 60927 to 61566 of RefSeqGene ID: NG_028016.3. In each of the following examples, the same region was used as the substrate double-stranded DNA.
  • Example 4 Polypeptide of the present invention, gRNA, and dissociation of substrate DNA 450 ng of the purified polypeptide (Cas12a-N or Cas12a-C) and 2 pmol guide RNA mixed, heated at 37 ° C. for 5 minutes, after the reaction 4 °C. 100 ng of the substrate double-stranded DNA (SEQ ID NO: 4) was mixed with the reaction solution and heated at 37° C. for 20 minutes. After the reaction, EDTA was added to a final concentration of 16.7 mM, and the polypeptide, gRNA, and substrate DNA of the present invention were divided into (1) a system heated at 95°C for 5 minutes and (2) a system not heated. dissociation was tested. After the reaction, the temperature was kept at 4°C. A portion of the reaction solution was then subjected to agarose gel electrophoresis.
  • the results show that the complex of the polypeptide of the present invention and guide RNA is very stable, and binds to the target double-stranded DNA while forming the complex until EDTA addition and high-temperature treatment. rice field.
  • Example 5 Heat resistance 450 ng of the above purified polypeptide (Cas12a-N or Cas12a-C) and 2 pmol of guide RNA (SEQ ID NO: 3) were mixed, and each temperature (37, 45, 55, 65, 75, 85, 95 ° C.) and heated for 5 minutes. Next, 100 ng of a substrate double-stranded DNA (SEQ ID NO: 4) was added to the mixed solution after heating, and the mixture was heated at 37°C for 1 hour. After that, EDTA was added so that the final concentration was 16.7 mM, and the mixture was heated at 95°C for 5 minutes. A portion of the reaction solution was subjected to agarose gel electrophoresis to examine the presence or absence of cleavage of the substrate DNA.
  • Example 6 Temperature Optimum 450 ng of the above purified polypeptide (Cas12a-N or Cas12a-C) and 2 pmol of guide RNA (SEQ ID NO:3) were mixed and heated at 37° C. for 5 minutes. Then, to a solution containing 100 ng of substrate double-stranded DNA (SEQ ID NO: 4) heated in advance at each temperature (37, 45, 55, 65, 75, 85, 95 ° C.) for 20 minutes, The mixture was added and maintained at that temperature for 20 minutes. After that, EDTA was added so that the final concentration was 16.7 mM, and the mixture was heated at 95°C for 5 minutes. A portion of the reaction solution was then subjected to agarose gel electrophoresis.
  • SEQ ID NO:3 2 pmol of guide RNA
  • Example 7 Length of guide RNA 450 ng of the purified polypeptide (Cas12a-N or Cas12a-C) and 2 pmol of guide RNA with different target recognition sequence chain lengths were mixed and heated at 37°C for 5 minutes. Next, a solution containing 100 ng of substrate double-stranded DNA (SEQ ID NO: 4) preheated to 37° C. was added to the heated mixed solution, and the mixture was heated at 65° C. for 20 minutes. Then, EDTA was added to a final concentration of 16.7 mM and heated at 95°C for 5 minutes to inactivate the polypeptide. A portion of the reaction solution was then subjected to agarose gel electrophoresis.
  • SEQ ID NO: 4 substrate double-stranded DNA
  • the guide RNA includes the base sequence of SEQ ID NO: 3 (corresponding to 23b in FIG. 5), and 8 types of guide RNA (from 22b in FIG. 15b) was prepared and used.
  • Example 8 Cleavage of single-stranded DNA 900 ng of the above purified polypeptide (Cas12a-N or Cas12a-C), 4 pmol of guide RNA (SEQ ID NO:3), 200 ng of double-stranded DNA (SEQ ID NO:4), and 5 pmol of single strand DNA (SEQ ID NO: 5) was mixed and heated at 65°C for 60 minutes. A fluorescent dye FAM is bound to the 5′ end of the target single-stranded DNA, and a quenching group BHQ1 is bound to the 3′ end. Trademark) Real Time System III (Takara Bio Inc.).
  • Example 9 PAM Sequence A PAM library having a sequence of "NNNNN" in the 5' upstream region of the target sequence human DNMT1 gene was constructed in a plasmid. The library was digested under the same conditions as in Example 7. An adapter for MGI sequencer was added to the resulting cleavage product, and the contained PAM sequence was subjected to nucleotide sequence analysis using the sequencer (MGI). In addition, the sequences of the PAM library that had not been cleaved were also subjected to nucleotide sequence analysis using the sequencer, and the obtained reads were used for normalization.
  • Example 10 Preparation of Dead Cas Polypeptide (1) Construction of Mutant Plasmid A mutant (SEQ ID NO: 7) was artificially synthesized in which bases gat (encoding aspartic acid) at positions 2833-2835 of SEQ ID NO: 2 were replaced with gcc (encoding alanine).
  • the nucleic acid of SEQ ID NO: 7 was inserted into the in-fusion cloning site of the expression vector pET6xHN-N (Takara Bio USA) according to the instruction manual attached to the kit. Escherichia coli BL21 DE3 strain was transformed with the resulting plasmid, and ampicillin-resistant clones were selected. From this clone was obtained the plasmid pET-Casl2a(D945A)-C for expression of the polypeptides of the invention.
  • the expression vector pET-Cas12a(D945A)-C has a structure in which a His tag is added to the C-terminal side of the expressed protein.
  • the dead Cas polypeptide was hereinafter referred to as Casl2a(D945A)-C.
  • Example 11 Nuclease activity loss of dead Cas polypeptide (1) 450 ng of the above purified Cas12a(D945A)-C and 2 pmol of guide RNA (SEQ ID NO:3) were mixed and heated at 37° C. for 5 minutes. After cooling to 4° C., 100 ng of double-stranded DNA (SEQ ID NO: 4) was added and heated at 65° C. for 60 minutes. After that, EDTA was added so that the final concentration was 16.7 mM, and the mixture was heated at 95°C for 5 minutes. A portion of the reaction solution was then subjected to agarose gel electrophoresis.
  • Example 12 Nuclease activity loss of dead Cas polypeptide (2) 900 ng of the purified Casl2a(D945A)-C, 4 pmol of guide RNA (SEQ ID NO: 3), 200 ng of double-stranded DNA (SEQ ID NO: 4), and 5 pmol of target single-stranded DNA (SEQ ID NO: 5) are mixed, Heated at 65° C. for 60 minutes. A fluorescent dye FAM is bound to the 5′ end of the target single-stranded DNA, and a quenching group BHQ1 is bound to the 3′ end. Measured with Real Time System III (registered trademark) (Takara Bio Inc.). In parallel with this, the same measurements as in Example 8 were performed.
  • Real Time System III registered trademark
  • Example 13 Specific detection of target genes (1) Two-step method Target ⁇ RNA (0, 1, 5, 10, 50, or 100 copies, respectively), 5.2 ⁇ M ⁇ primer set 4, 1.4 mM each dNTPs, 65 U Bca DNA polymerase (Takara Bio Inc.), 1U of Reverse Transcriptase XL (AMV) (Takara Bio Inc.) and a buffer solution were mixed and heated at 63° C. for 30 minutes (RT-LAMP method).
  • the ⁇ primer set 4 is a mixture of FIP primer, BIP primer, LOOP-F primer, LOOP-B primer, F3 primer and B3 primer.
  • Reaction solution A was added to the amplified product of the RT-LAMP method and heated at 65°C for 60 minutes.
  • a fluorescent dye ROX is bound to the 5' end of the reporter nucleic acid, and a quenching group BHQ1 is bound to the 3' end of the reporter nucleic acid.
  • III (Takara Bio Inc.).
  • 5.2 ⁇ M ⁇ primer set 4 1.4 mM each dNTPs, 65 U Bca DNA polymerase (Takara Bio Inc.), 1 U Reverse Transcriptase XL (AMV) (Takara Bio Inc.), 2 U inorganic pyrophosphatase (PPase ) (Takara Bio Inc.), 5 pmol of a reporter nucleic acid (single-stranded DNA) (sequence: CCCCCC), and a buffer were mixed to prepare a reaction solution B.
  • Target ⁇ RNA (0, 1, 5, 10, 50, or 100 copies, respectively) and 4 ⁇ L of RNP were mixed with 14 ⁇ L of reaction solution B and heated at 65° C. for 60 minutes.
  • the 5′ end of the reporter nucleic acid is bound to the fluorescent dye FAM, and the 3′ end is bound to the quenching group BHQ1. III (Takara Bio Inc.).
  • thermostable Cas nuclease polypeptide of the present invention.
  • the thermostable Cas nuclease is useful in a wide range of fields such as genetic engineering, biology, medicine and agriculture.
  • thermostable Cas12a SEQ ID NO: 1 The amino acid sequence of thermostable Cas12a SEQ ID NO: 2: The nucleotide sequence of thermostable Cas12a SEQ ID NO: 3: The nucleotide sequence of guide RNA SEQ ID NO: 4: The nucleotide sequence of target double-strand DNA (human DNMT1) SEQ ID NO: 5: The nucleotide sequence of target single strand DNA SEQ ID NO: 6: The amino acid sequence of thermostable dead Cas12a SEQ ID NO: 7: The nucleotide sequence of thermostable dead Cas12a SEQ ID NO: 8: The nucleotide sequence of guide RNA SEQ ID NO: 9: The nucleotide sequence of reporter nucleic acid

Abstract

L'invention concerne une nouvelle nucléase Cas résistante à la chaleur appartenant à la classe 2 et du type V ; et un procédé de production y relatif.
PCT/JP2022/015608 2021-03-30 2022-03-29 Nouveau polypeptide ayant la capacité de former un complexe avec un arn guide WO2022210748A1 (fr)

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Citations (2)

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WO2018013990A1 (fr) * 2016-07-15 2018-01-18 Zymergen Inc. Assemblage d'adn et édition du génome sans cicatrice utilisant crispr/cpf1 et une adn ligase
WO2021041846A1 (fr) * 2019-08-30 2021-03-04 Inari Agriculture, Inc. Nucléases guidées par arn et protéines de liaison à l'adn

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WO2018013990A1 (fr) * 2016-07-15 2018-01-18 Zymergen Inc. Assemblage d'adn et édition du génome sans cicatrice utilisant crispr/cpf1 et une adn ligase
WO2021041846A1 (fr) * 2019-08-30 2021-03-04 Inari Agriculture, Inc. Nucléases guidées par arn et protéines de liaison à l'adn

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