WO2021258580A1 - Procédé de clonage in vitro de grands fragments d'adn à base de crispr/cas12a et applications associées - Google Patents

Procédé de clonage in vitro de grands fragments d'adn à base de crispr/cas12a et applications associées Download PDF

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WO2021258580A1
WO2021258580A1 PCT/CN2020/120332 CN2020120332W WO2021258580A1 WO 2021258580 A1 WO2021258580 A1 WO 2021258580A1 CN 2020120332 W CN2020120332 W CN 2020120332W WO 2021258580 A1 WO2021258580 A1 WO 2021258580A1
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cas12a
fragment
vector
crrna
dna
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张立新
谭高翼
梁敏东
王为善
刘乐诗
曾晓倩
李源航
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华东理工大学
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Definitions

  • the present invention relates to the field of molecular biology, in particular to a CRISPR/Cas12a-based in vitro (in vitro) large-segment DNA cloning method, kit and application thereof.
  • BGC hidden secondary metabolite biosynthetic gene clusters
  • the commonly used cloning methods mainly include the following categories: 1) Genomic library; 2) Red/ET and ExoCET systems based on recombinase; 3) Gibson assembly; 4) Utilization TAR technology of yeast recombination system; and 5) CRIPR/Cas9-mediated CATCH, etc. (see Table 1 below).
  • genomic library is not only complicated, time-consuming, and laborious in construction and screening, but also the target DNA fragments are often scattered on several different clones. It is often necessary to subclone and delete them when conducting gene function research. The extra sequence may need to be stitched into a complete biosynthetic pathway.
  • Rec/ET cloning technology The principle of Rec/ET cloning technology is: Rac phage recombinant protein, namely full-length RecE and RecT, can efficiently mediate homologous recombination (line-line recombination) between linear DNA molecules in E. coli cells.
  • RecE is a 5'-3' exonuclease
  • RecT is a single-stranded DNA annealing protein
  • the protein-protein interaction between RecE and RecT is necessary for linear recombination
  • the linear recombination of Rec/ET combined action The efficiency is 1000 times that of a single action.
  • the Rec/ET cloning technology is difficult to clone DNA fragments larger than 50kb from the bacterial genome, nor can it clone DNA fragments from the mammalian genome. This is because the Rec/ET cloning technology relies on the Rec/ET recombinase expressed in the cell. Only when the cloning vector and the target DNA fragment enter and meet in an E. coli cell at the same time can homologous recombination occur.
  • ExoCET cloning technology was further developed: in vitro the genomic DNA and cloning vector were treated with exonuclease, and then the in vitro reaction products were subjected to homologous recombination in the presence of Rec/ET recombinase, thereby Established ExoCET cloning technology.
  • ExoCET technology can clone DNA fragments >100kb directly from the bacterial genome, and can clone DNA fragments >50kb from mammalian cells and human blood.
  • ExoCET technology can also assemble at least 20 DNA fragments to form a complete plasmid. However, since this technology is based on gene recombination for cloning, mismatches may occur for high GC fragments and repetitive sequences.
  • CATCH Cas9-Assisted Targeting of Chromosome Segments
  • Cas9 in vitro cutting and Gibson assembly uses Cas9 in vitro cutting and Gibson assembly to clone large DNA fragments onto BAC.
  • This method is currently only applied to prokaryotic genomes, and PCR pre-screening of colonies is required before restriction enzyme digestion of recombinant DNA is performed.
  • the combined in vitro recombination technology of CRISPR and Gibson technology can only effectively clone DNA fragments below 100kb.
  • gene editing technology can also be combined with bacterial Red/ET cloning technology to clone large fragments of DNA.
  • Baker et al transformed DNA fragments cleaved by CRISPR and linear vectors into bacteria expressing the lambda phage Red/ET recombination system.
  • the DNA fragments can be assembled into a single recombinant in the bacteria through a sequence-dependent enzymatic reaction.
  • the Red/ET cloning technology it is theoretically difficult to obtain a single recombinant of more than 50kb.
  • BAC bacterial artificial chromosome
  • ULCC upper limit of cloning ability
  • BGC biosynthetic gene cluster
  • NP natural product
  • CATCH Cas9 assisted chromosome segment targeting
  • TAR transformation-related recombination
  • YA yeast assembly
  • GA Gibson assembly
  • ExoCET Exonuclease combined with RecET recombination.
  • CRISPR/Cas12a is a single RNA guided (crRNA) endonuclease of the class II CRISPR/Cas system.
  • Cas12a recognizes T-rich protospacer-motif (PAM) instead of Cas9's G-rich PAM, and produces sticky ends, which has a wide range of application prospects.
  • PAM protospacer-motif
  • CRISPR/Cas12a is also widely used in nucleic acid-based diagnostic applications, small molecule detection, etc.
  • CRISPR/Cas12a has obvious advantages in DNA assembly.
  • Wang Jing’s team was the first to report C-Brick, a modular assembly method based on CRISPR/Cas12a DNA sequence, which realized the assembly and expression of three pigment protein genes; subsequently, they reported the DNA in vitro group transfer method-CCTL, Complete the replacement of the promoter of actinomycin gene cluster and greatly increase its yield.
  • CRISPR/Cas12a-based cloning technology for large fragments of DNA in vitro.
  • the present invention provides a CRISPR/Cas12a-based in vitro large fragment DNA cloning method for cloning large DNA fragments.
  • This method can easily, quickly and efficiently clone large fragments of DNA (for example, >10kb, or even >100kb) from DNA samples (for example, genome); more surprisingly, this method has a higher G+C content
  • the cloning of large fragments of DNA (>60%) is particularly effective.
  • the present invention combines the CRISPR/Cas12a system with a cloning vector, through complementary binding between the sticky end generated at or near the end of the target DNA fragment and the homology arm formed at the end of the target DNA fragment.
  • the sticky ends are combined, so that large fragments of DNA can be cloned efficiently and ensure the integrity of large fragments of DNA.
  • the present invention provides an in vitro large-segment DNA cloning method based on the CRISPR/Cas12a system, the method comprising:
  • (1) Construction and cutting of the capture vector prepare homology arms at both ends of the target DNA fragment, and connect the homology arms to the vector to obtain the capture vector, wherein the homology arm contains at least one A PAM site that can be recognized by Cas12a or its biologically active functional fragment or variant; based on the crRNA at least partially complementary binding to the homology arm, the Cas12a or its biologically active functional fragment or variant is used for the The capture carrier is cut to obtain a cut capture carrier;
  • target DNA fragments based on the crRNA and using the Cas12a or its biologically active functional fragment or variant, the sample containing the target DNA fragment is cut to obtain the target DNA fragment;
  • the cut capture vector is ligated with the target DNA fragment, and transferred/introduced into a host cell to obtain a recombinant host cell with the target DNA fragment.
  • the sample is an isolated nucleic acid sample, such as an isolated DNA sample.
  • the sample may be a genome and/or a metagenomic group, or a DNA sample derived from a genome and/or metagenomic group (for example, a DNA library, including a BAC library and a YAC library).
  • the cloning method of the present invention is used to clone a biosynthetic gene cluster.
  • the biosynthetic gene cluster can be predicted by antiSMASH.
  • the present invention provides an in vitro biosynthetic gene cluster (BGC) cloning method based on the CRISPR/Cas12a system, the method comprising:
  • BGC prediction predict BGC through online tools
  • (2) Construction and cutting of the capture vector prepare the homology arms at both ends of the BGC, and connect the homology arms to the vector to obtain the capture vector, wherein the homology arms include at least one A PAM site that can be recognized by Cas12a or its biologically active functional fragment or variant; based on the crRNA at least partially complementary binding to the homology arm, the Cas12a or its biologically active functional fragment or variant is used for the The capture carrier is cut to obtain a cut capture carrier;
  • the cut capture vector is connected to the BGC, and transferred/introduced into a host cell to obtain a recombinant host cell with the BGC.
  • the sample is an actinomycete genome.
  • the sample is a Streptomyces genome.
  • the present invention also provides a method for improving the cutting efficiency of the CRISPR/Cas12a system, the method comprising in a hydrochloride buffer at a temperature of 35°C-38°C, preferably 37°C, for 40min-120min , Preferably 50min-100min, more preferably 60min-80min.
  • the present invention also provides a kit for cloning large fragments of DNA in vitro, the kit comprising a vector, Cas12a or its biologically active functional fragment or variant, or expressing the Cas12a or its biologically active functional fragment or variant.
  • Expression vector and instructions for use.
  • the kit may further include a buffer for CRISPR/Cas12a cleavage, and the buffer is a hydrochloride buffer with a pH of 7.5-8.0, preferably 7.9.
  • the present invention relates to in vitro large-segment DNA cloning methods, kits and related applications mediated by the CRISPR/Cas12a system. Its advantage is that it can quickly clone large-segment DNA with high GC content, and its operation is simple and does not rely on expensive equipment. , Save time and cost, and have a high positive rate and have the potential for market application.
  • the cloning method of the present invention can also be used in the genome mining method of biosynthetic gene cluster (BGC), it is also named CAT-FISHING (CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning platform). This method does not use restriction enzymes to randomly cut the genome, but uses Cas12 and targeted crRNA to accurately cut target fragments.
  • CAT-FISHING will become a simpler and more effective method for in vitro direct cloning of large BGCs with high GC.
  • Fig. 1 is a schematic diagram of the working principle of the cloning method according to the present invention.
  • LHA left homology arm
  • RHA right homology arm
  • BGC biosynthetic gene cluster.
  • FIG. 2 shows the optimized results of Cas12a digestion system buffer.
  • Figure 3 shows the optimization result of Cas12a digestion system reaction time.
  • Figure 4 shows the optimized result of the molar ratio of the Cas12a PCR product to the Cas12a digestion system.
  • Figure 5 is a flow chart of the cloning efficiency of the cloning method and NEB restriction endonuclease method according to the present invention.
  • Figure 6 shows the PCR verification and sequencing of transformants obtained according to the cloning method of the present invention.
  • A Schematic diagram of PCR screening.
  • B PCR results of ten randomly selected clones. "-" indicates a blank control, using the genome of E. coli DH10B as a PCR template.
  • C Schematic diagram of crRNA design and sticky end joining. Using a crRNA with an 18 nt spacer sequence, Cas12a cuts mainly after the 14th base, resulting in a sticky end of 8 nt.
  • D Sequencing result of vector-fragment junction.
  • Figure 7 is a comparison of the number of clones and the accuracy of the cloning method according to the present invention and the NEB restriction enzyme method.
  • Figure 8 shows the optimized result of 50kb-BAC-up/dn-crRNA.
  • Figure 9 shows the cloning of large DNA fragments of different lengths from BAC plasmids.
  • A Diagram of the 50kb and 80kb target fragments in the BAC plasmid.
  • B Pulse field gel electrophoresis (PFGE) results of BAC plasmid digested with CRISPR/Cas12a.
  • PFGE Pulse field gel electrophoresis
  • Figure 10 shows that the correct clone containing pBAC2015-50kb-BAC was screened by PCR.
  • A Schematic diagram of pBAC2015-50kb-BAC plasmid junction and PCR products.
  • F1, F2, and F3 are PCR products, using 50-BAC-scr-up-F/R, 50-BAC-scr-middle-F/R and BAC-scr-down-F/R (also known as BAC- scr-dn-F/R) primers for amplification.
  • B, C, and D are the results of PCR amplification. In three independent repeated experiments, 12 random clones were screened for PCR verification.
  • Figure 11 shows that the correct clone containing pBAC2015-80kb-BAC was screened by PCR.
  • A Schematic diagram of pBAC2015-80kb-BAC plasmid junction and PCR products.
  • F1, F2, and F3 are PCR products, which are expressed as 80-BAC-scr-up-F/R, 80-BAC-scr-middle-F/R and 80-scr-down-F/R (also known as BAC- scr-dn-F/R) primers for amplification.
  • B, C, and D are the results of PCR amplification. In three independent repeated experiments, 12 random clones were screened for PCR verification.
  • Figure 12 shows the result of restriction digestion of the recombinant plasmid.
  • A pBAC2015-50kb-BAC restriction digestion verification result.
  • B pBAC2015-80kb-BAC restriction digestion verification result.
  • Figure 13 shows the number of clones and positive rates of the 50kb and 80kb target fragments.
  • Fig. 14 shows the operation flow of in vitro cloning of the target BGC by the method according to the present invention.
  • Figure 15 shows restriction endonuclease verification of five randomly selected positive clones.
  • A Paulomycin gene cluster cloned recombinant plasmid XhoI restriction map.
  • B SmLI restriction map of the recombinant plasmid cloned from the Surugamides gene cluster. The restriction band is indicated by the arrow.
  • target DNA fragment refers to a target DNA fragment that needs to be cloned, which can be a genomic fragment or an artificially synthesized exogenous fragment, or a complete gene.
  • genome includes naturally occurring genomes and synthetic genomes, and includes genetically modified genomes, such as genomes that did not exist in the laboratory and in nature before, including modified genomes and containing nucleic acids and/or parts from more than one species Hybrid genome of genome.
  • the term “genome” includes organelle genomes (eg, mitochondrial and chloroplast genomes), genomes of self-replicating organisms (cell genomes), which include prokaryotic and eukaryotic organisms, fungi, yeast, bacteria (e.g., mycoplasma), archaea, vertebrates , Mammalian and other biological genomes, viral genomes, and other genomes that rely on the host to multiply.
  • the genome also includes those of organisms and synthetic organisms that do not fall into any known Linnean classification.
  • Exemplary genomes can be microbial genomes, such as the genomes of single-celled organisms including bacteria and yeast.
  • ⁇ (Metagenome) (also known as Microbial Environmental Genome, or metagenome) refers to the sum of the genetic material of all micro organisms in the environment. It contains the genes of cultivable and unculturable microorganisms, and mainly refers to the sum of the genomes of bacteria and fungi in environmental samples.
  • the term "large fragment DNA” refers to a DNA molecule with a length of 10 kb or more, for example, a length of 20 kb or more, 30 kb or more, 40 kb or more, 50 kb or more, 60 kb or more, 70 kb or more, 80 kb or more, 90 kb or more, 100 kb in length.
  • high GC content means that the G+C content in the nucleic acid molecule is higher than 60%, for example, 65% or higher, including 65%, 70%, 75%, or 80%.
  • vector refers to a vector that can be assembled with or inserted into an exogenous fragment, and usually contains an origin of replication and nucleic acid sequences of other entities necessary for replication and/or maintenance in a host cell. .
  • the vector can be a circular plasmid or a linear vector, such as, but not limited to, a host-specific plasmid, a shuttle plasmid, a cosmid, a bacterial artificial chromosome (BAC) or a yeast artificial chromosome (YAC).
  • the vector can also be a cloning vector or an expression vector.
  • a host-specific plasmid the vector can only be used in Escherichia coli, Streptomyces, Bacillus subtilis, Corynebacterium glutamicum, fungi (for example, Plasmids replicated in Saccharomyces cerevisiae, S. pombe, Pichia membranaefaciens, and mammalian cells.
  • the vector may be a shuttle plasmid, such as an Escherichia coli-Saccharomyces cerevisiae shuttle plasmid, an Escherichia coli-Streptomyces shuttle plasmid, an Escherichia coli-Bacillus subtilis shuttle plasmid, an Escherichia coli-Corynebacterium glutamicum shuttle plasmid, a bacteria-mammalian cell Shuttle plasmid, bacteria-plant cell shuttle plasmid, Escherichia coli-filamentous fungus shuttle plasmid, Escherichia coli-Streptomyces-Saccharomyces cerevisiae shuttle plasmid, Escherichia coli-filamentous fungus-Saccharomyces cerevisiae shuttle plasmid, Escherichia coli-filamentous fungus-Saccharomyces cerevisiae shuttle plasmid, Escherichia
  • the filamentous fungi include, but are not limited to, Aspergillus niger, Aspergillus oryzae, and Aspergillus flavus.
  • bacterial artificial chromosomes BAC
  • yeast artificial chromosomes YAC
  • the carrier may be a carrier known in the art.
  • the vector can accommodate large fragments of DNA, such as pCC2FOS (from EpicentreBio) or pBAC2015 (Wang H et al., Nature Protocols, 2016, 11(7): 1175-1190.).
  • identity is calculated by comparing two aligned sequences in a comparison window.
  • the alignment of the sequences makes it possible to determine the number of positions (nucleotides or amino acids) shared by the two sequences in the comparison window. Then, divide the number of common positions by the total number of positions in the comparison window and multiply by 100 to obtain the percent homology.
  • the determination of the percent sequence identity can be done manually or using a well-known computer program.
  • the term “complementary” refers to the hydrogen bond base pairing between the nucleotide bases G, A, T, C and U to form a preference hierarchy (hierarchy) so that when two given polynucleotides When acid or polynucleotide sequences anneal to each other, A pairs with T and G pairs with C in DNA, and G pairs with C and A pairs with U in RNA.
  • substantially complementary means that the nucleic acid molecule or part thereof (for example, a primer) has at least 90% complementarity, for example, 90%, to the second nucleotide sequence over the entire length of the molecule or part thereof. Complementary, 95% complementary, 98% complementary, 99% complementary or 100% complementary.
  • operably linked means that the promoter is in the correct functional location and/or orientation relative to the nucleic acid sequence regulated by the promoter (to control the transcription initiation and/or expression of the sequence).
  • nucleic acid or polypeptide refers to a nucleic acid or polypeptide that is separated from at least one other component (for example, nucleic acid or polypeptide) that is in its natural state.
  • the source is present with the nucleic acid or polypeptide, and/or when expressed by the cell or secreted by the cell in the case of a secreted polypeptide, the component will be present with the nucleic acid or polypeptide.
  • Chemically synthesized nucleic acids or polypeptides or nucleic acids or polypeptides synthesized using in vitro transcription/translation are considered "isolated".
  • left homology arm and “upstream homology arm” can be used interchangeably; the terms “right homology arm” and “downstream homology arm” can be used interchangeably.
  • the large-segment DNA cloning method of the present invention is based on the complementary binding between the sticky ends produced by Cas12a/crRNA, which makes it possible to improve the efficiency of large-segment DNA cloning (that is, the cloning time is short and the positive High rate), while ensuring the integrity of the target DNA fragment.
  • the method of the present invention shows similar high-efficiency cloning.
  • the present invention provides an in vitro large-segment DNA cloning method based on the CRISPR/Cas12a system, the method comprising:
  • (1) Construction and cutting of the capture vector prepare homology arms at both ends of the target DNA fragment, and connect the homology arms to the vector to obtain the capture vector, wherein the homology arm contains at least one A PAM site that can be recognized by Cas12a or its biologically active functional fragment or variant; based on the crRNA at least partially complementary binding to the homology arm, the Cas12a or its biologically active functional fragment or variant is used for the The capture carrier is cut to obtain a cut capture carrier;
  • target DNA fragments based on the crRNA and using the Cas12a or its biologically active functional fragment or variant, the sample containing the target DNA fragment is cut to obtain the target DNA fragment;
  • the cut capture vector is ligated with the target DNA fragment, and transferred/introduced into a host cell to obtain a recombinant host cell with the target DNA fragment.
  • the homology arm is at least 100 bp away from the 5'end or 3'end of the target DNA fragment, preferably at least 200 bp, more preferably at least 500 bp, further preferably at least 1 kb, more preferably at least 1.2 kb, more preferably At least 1.5kb. That is to say, taking the upstream homology arm as an example, its 3'end is at least 100 bp away from the 5'end of the target DNA fragment, preferably at least 200 bp, more preferably at least 500 bp, further preferably at least 1 kb, more preferably at least 1.2 kb, and more preferably at least 1.5kb.
  • the selection of homology arms can be non-restrictively considering the selection of abundant sequences in PAM sites (TTTN), which provides more options for crRNA.
  • the length of the homology arm is more than 100 bp.
  • the length of the homology arm is 200bp or more, 300bp or more, 400bp or more, 500bp or more, 600bp or more, 700bp or more, 800bp or more, 900bp or more, 1000bp or more, 1100bp or more, 1200bp or more, 1300bp or more, 1400bp or more, or Above 1500bp.
  • the length of the homology arm is 800bp-1500bp, preferably 800bp-1200bp, more preferably 900bp-1200bp.
  • the homology arm includes 1, 2, 3, 4, 5 or more PAM sites that can be recognized by Cas12a or a biologically active functional fragment or variant thereof.
  • the homology arm contains 2 or 3 PAM sites that can be recognized by Cas12a or its biologically active functional fragment or variant.
  • the homology arms at both ends of the target DNA fragment may be located within the target DNA fragment, outside the target DNA fragment, or include the 5'end or 3'end of the target DNA fragment.
  • the homology arms at both ends of the target DNA fragment are located outside the target DNA fragment.
  • one of the purposes of the homology arm is to form a sticky end that can complementarily bind to the sticky ends adjacent to the 5'and 3'ends of the target DNA fragment. Therefore, the homology arm only needs to include adjacent
  • the sequence at the 5'and 3'ends of the target DNA fragment may be the sequence required for cleavage by the Cas12a enzyme, which includes a sequence that is at least partially complementary (for example, 70% or more) binding to the crRNA, PAM sequence, and Cas12a enzyme The sequence of cutting, and the sequence between them.
  • the corresponding 5'and/or 3'homology arms are in addition to It is intended to generate complementary sticky ends and also include the terminal sequence of the target DNA fragment, so that the obtained target DNA fragment is complete.
  • the vector used to connect the homology arms can be determined based on factors such as the length of the specifically cloned DNA fragment, the host cell used, and subsequent operations (whether to perform expression, etc.).
  • the vector used to prepare the capture vector is a circular plasmid or a linear vector.
  • the capture vector is selected from plasmids, cosmids, bacterial artificial chromosomes (BAC) or yeast artificial chromosomes (YAC).
  • BAC vector, or a variant thereof can be selected, such as the pBAC2015 vector.
  • a specific shuttle vector can be selected.
  • the homology arms can be connected to the vector by techniques known in the art (such as homologous recombination or restriction ligation, such as Gibson ligation).
  • the CRISPR/Cas system is divided into two categories: class 1 CRISPR/Cas system uses multiple effector protein complexes to interfere with target genes;
  • the class2 CRISPR/Cas system uses a single nuclease-nucleic acid complex to cut exogenous nucleic acids, thereby resisting external invasion.
  • the class2 system is simple and efficient, so it has received widespread attention.
  • Typical representatives of the 2 types of CRISPR/Cas systems include Cas9, Cas12a (Cpf1) and Cas13, etc., which have now been successfully applied in the fields of DNA editing, gene regulation, and small molecule detection.
  • the designed Cas12a can recognize more PAM sequences, such as 5'-TYCV, 5'-TATV.
  • Cas12a can cut RNA, process pre-crRNA to produce mature crRNA, and use the RNA to cut target genes without the participation of RNase III.
  • Cas12a only needs one crRNA to cut the target gene without tracrRNA.
  • Cas12a cuts dsDNA to form sticky ends, which is conducive to DNA insertion.
  • the Cas12a cleavage site is far away from the recognition site, which provides more space for gene editing.
  • the CRISPR enzyme in the CRISPR/Cas system can interact with gRNA to form a gRNA-CRISPR enzyme complex, that is, a CRISPR complex, and can allow the guide sequence to approach the target sequence containing the PAM sequence under the cooperation of the gRNA.
  • gRNA guide nucleic acid
  • crRNA crRNA
  • the PAM sequence is a sequence present in the target gene or nucleic acid, which can be recognized by the class II CRISPR enzyme.
  • the PAM sequence can be changed according to the source of the type II CRISPR enzyme. That is, depending on the species, there are different PAM sequences that can be specifically recognized.
  • the PAM sequence recognized by the Cas12a enzyme can also be 5'-TTT/N-3' (N is A, T, C, or G).
  • N is A, T, C, or G.
  • PAM may be changed as research progresses on mutants of the source of the enzyme.
  • the Cas12a enzyme can be a Cas12a enzyme derived from: Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Neisseria Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus , Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Fengyou bacteria ( Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methy
  • the biologically active functional fragment or variant of Cas12a refers to the biologically active fragment, variant, or fusion protein of the naturally-occurring Cas12a polypeptide, which contains at least 80%, 85%, and preferably at least 90% of the naturally-occurring Cas12a polypeptide. , 95%, 97%, 98%, 99%, or 100% identical amino acid sequences.
  • fragment or variant is understood to include biologically active fragments or biologically active variants that exhibit "biological activity” as described herein. That is, the biologically active fragment or variant of Cas12a exhibits biological activity that can be measured and tested.
  • a biologically active fragment or variant exhibits the same or substantially the same biological activity as the natural (ie wild or normal) Cas12a protein, and such biological activity can be evaluated by the fragment or variant, for example, in terms of cleaving the target DNA sequence.
  • the variant of the CRISPR enzyme may further include optional functional domains.
  • the CRISPR enzyme mutant may have additional functions in addition to the original functions of the wild-type CRISPR enzyme.
  • the functional domain can be a domain having methylase activity, demethylase activity, histone modification activity, RNA cleavage activity, or nucleic acid binding activity, or a tag or label for separating and purifying proteins (including peptides). Reporter gene, but the present invention is not limited to this.
  • Tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags and thioredoxin (Trx) tags; reporter genes include glutathione- S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), ⁇ -galactosidase, ⁇ -glucuronidase, luciferase, autofluorescent protein (Including green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP)), but the present invention is not limited thereto.
  • the crRNA used herein refers to target DNA-specific RNA, which can form a complex with the Cas12a protein and guide the Cas12a protein to the target sequence.
  • a crRNA can contain multiple domains. Each domain may have intra-strand or inter-strand interactions of gRNA in a three-dimensional form or an active form.
  • the crRNA includes a spacer sequence and a binding sequence of the Cas12a protein.
  • the crRNA may be composed of 5'-[Cas12 protein binding sequence]-[spacer sequence]-3', but the present invention is not limited thereto.
  • there is a linker sequence between the binding sequence and the spacer sequence and the length of the linker sequence is 1-15 bp, preferably 2-10 bp.
  • the binding sequence of the Cas12a protein can be the binding sequence of the Cas12a protein contained in the naturally-occurring species, can be derived from the binding sequence of the Cas12a protein contained in the naturally-occurring species, or can be combined with the Cas12a protein contained in the naturally-occurring species
  • the binding sequence has partial or complete homology.
  • the binding sequence of Cas12a protein can be 16-32, 18-25, more preferably 20-21, most preferably 21 base sequence, or 16-32, 18-25, more preferably 20-21 A sequence of 21 bases is most preferred.
  • the binding sequence of the Cas12a protein can have partial (ie at least 50% or more) or complete homology with the binding sequence of the Cas12a protein of the following bacteria or the binding sequence of the Cas12a protein derived therefrom: Parcubacteria bacterium (GWC2011_GWC2_44_17) ), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp.
  • BV3L6 Porphyromonas macacae (Porphyromonas macacae), 2006 ), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC_KO8D17), Leptospira (Leptospira ininadai), Laevis (MA2020), New Murder Francis (Francisella novicida) (U112), Candidatus Methanoplasma termitum or Eubacterium eligens.
  • the spacer sequence may be a nucleic acid sequence that has complementarity with the target sequence, for example, has at least 70%, 75%, 80%, 85%, 90%, or 95% or higher (e.g., 100%) complementarity or complete complementarity.
  • the spacer sequence can be 15-50, preferably 16-25, more preferably 17-19, most preferably 18 base sequence, or contains 15-50, preferably 16-25, more preferably 17-19 , Most preferably a sequence of 18 bases.
  • the spacer sequence is preferably 17-19, such as 17, 18 or 19 bases; in a further preferred embodiment, the spacer sequence is preferably 18 bases.
  • the spacer sequence may have 0-5 mismatch binding to the target sequence.
  • the spacer sequence may have 0, 1, 2, 3, 4, or 5 mismatch bindings to the target sequence.
  • the target sequence may be a base sequence near the PAM sequence of the homology arm.
  • the spacer sequence is capable of at least partially complementary binding to a contiguous 15-50 bp, preferably 16-25 bp, more preferably 17-19 bp target nucleic acid sequence near the PAM in the homology arm.
  • the target sequence may be a contiguous 15-50, preferably 16-25, more preferably 17-19, most preferably 18 base sequence of the 3'end or 5'end of the PAM.
  • crRNA can be transcribed in vitro or artificially chemically synthesized.
  • the method for in vitro transcription and synthesis of crRNA is known in the art.
  • the crRNA primer template used for in vitro reverse transcription of crRNA can contain three parts, the 5'end is a spacer sequence that is reversely complementary to the target gene fragment, the middle is the binding sequence of the Cas12a protein, and the 3'end is the binding of the T7 promoter during in vitro transcription. sequence.
  • the selection of 5'spacer sequence is the key to crRNA design.
  • the guide sequence can be designed with the aid of crRNA design software (for example, CRISPR RGEN Tools).
  • crRNA with excellent cleavage effect can be selected based on the PAM on the homology arm.
  • the length of the target DNA fragment may be 10 kb or more, for example, 50 kb or more, 60 kb or more, 70 kb or more, 80 kb or more, 90 kb or more, 100 kb, 110 kb or more, 120 kb or more, 130 kb or more, or even 140 kb or more.
  • the length of the target DNA fragment may be 50 kb-140 kb.
  • the target DNA fragment may have a GC content of 60% or more (even a high GC content of 70% or more).
  • the target DNA fragment targeted by the cloning method of the present invention is not limited to its source. It can be a DNA fragment derived from a prokaryote or DNA fragments of eukaryotes. In some embodiments, it may be a DNA fragment that exists in a natural state, or a DNA fragment that exists in an unnatural state, for example, a synthetic or modified DNA fragment.
  • the target DNA fragment may be contained in the sample.
  • the sample may further include proteins, cells, fluids, biological fluids, protective agents, and/or other substances.
  • the sample is an isolated nucleic acid sample.
  • the isolated nucleic acid sample is an isolated DNA sample.
  • the sample is a genome and/or a metagenomic group, or a DNA sample derived from a genome and/or metagenomic group (for example, a DNA library, including a BAC library and a YAC library).
  • the sample may be a DNA library, genomic DNA, metagenomic DNA, or other artificial DNA, or a combination thereof.
  • the sample can be a phage-based library (e.g., lambda phage, P1 phage, and fosmid, etc.) and an artificial chromosome library (e.g., bacterial artificial chromosome (BAS) library, yeast artificial chromosome (YAC), P1 artificial Chromosome (PAC) library and mammalian artificial chromosome (MAC) library, etc.).
  • the sample may be a genomic library or a cDNA library.
  • the sample can be obtained from mammalian cells, viruses, bacteria, fungi, yeasts, protozoa, microorganisms, parasites, and the like.
  • the sample may be a bacterial genome, for example, an actinomycete genome.
  • the sample can be collected fresh. In some embodiments, the sample can be stored before being used in the methods and kits described herein. In some embodiments, the sample is an untreated sample. As used herein, "untreated sample” refers to a biological sample that has not been subjected to any prior sample pretreatment, except for being diluted in a solution and/or suspended in a solution. In some embodiments, samples can be obtained from mammalian cells, viruses, bacteria, fungi, yeast, protozoa, microorganisms, parasites, etc., and can be stored or stored before being used in the methods and kits described herein. Processing. By way of non-limiting example, the sample can be embedded in paraffin, refrigerated or frozen.
  • Frozen samples can be thawed before processing nucleic acids according to the methods and kits described herein.
  • the sample may be a processed or processed sample. Exemplary methods for processing or processing samples include, but are not limited to: centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, contact with protective agents (e.g., nuclease inhibitors), and any combination of the above methods .
  • the sample can be processed with chemical and/or biological reagents. Chemical reagents and/or biological reagents can be used to protect and/or maintain the stability of the sample or the nucleic acid contained in the sample during processing and/or storage. Alternatively or additionally, chemical and/or biological reagents can be used to release nucleic acid from other components of the sample. Those skilled in the art are familiar with sample processing, preservation or processing methods and procedures for nucleic acid processing and/or analysis.
  • the nucleic acid present in the sample can be separated, enriched, or purified before being used in the methods and kits described herein.
  • Methods for separating, enriching or purifying nucleic acids from samples are well known to those of ordinary skill in the art.
  • kits for isolating genomic DNA from various sample types are commercially available (e.g., catalog numbers 51104, 51304, 56504, and 56404; Qiagen; Germantown, MD).
  • the DNA sample can be embedded in low melting point agarose before or during the operation of the sample according to the method and kit of the present invention.
  • low-melting agarose By using low-melting agarose to embed DNA, mechanical shearing of large DNA sequences can be avoided.
  • the bacterial genomic DNA is separated by the following method: the bacteria from which the genome is to be extracted are embedded with low melting point agarose; then, the embedded bacteria are treated with lysozyme and proteinase K.
  • the bacteria from which the genome is to be extracted are freshly cultured bacteria.
  • the bacteria are Streptomyces, the bacteria are mycelium cultured aerobic at 30°C for 24h-30h.
  • the amount of bacteria in each embedding block is 4-5 mg, and lysozyme is lysed for 1 to 2 hours, which can shorten the proteinase K digestion time to 2 hours.
  • the sequence information of the target DNA fragment may be known or unknown.
  • the sequence at both ends of the target DNA is known, or can be inferred based on the known sequence information of a species that is closely related to the target DNA. For example, a DNA sample containing target DNA has complete sequencing information, or has partial sequencing information.
  • step (1) before cutting the capture carrier, a step of screening and separating the capture carrier is further included.
  • a selection marker can be introduced into a capture vector with a homology arm.
  • the selection markers are non-limitingly selected from: resistance selection markers, reverse selection genes (such as sacB gene), lacZ selection system reporter genes, and the like. Resistance markers are well known. The skilled person can determine suitable resistance markers for different host/donor combinations.
  • the selection marker is located between the homology arms, or the selection marker can be located on the vector.
  • the selection marker may be lacZ. In one embodiment, the lacZ may be located between the 5'and 3'homology arms.
  • the separation of most capture carriers can be performed using techniques known in the art.
  • the Cas12a/crRNA digestion system includes Cas12a protein and crRNA and optionally a suitable digestion buffer.
  • the Cas12a enzyme or its biologically active functional fragments or variants used in the present invention can be obtained commercially, or can be prepared by protein purification methods known in the art.
  • the Cas12a enzyme or its biologically active functional fragment or variant has the function of Cas12a enzyme to cut double-stranded DNA.
  • Cas12a may be LbCas12a.
  • the buffer used in the Cas12a/crRNA digestion system has a pH value of 7.5-8.0, for example, pH 7.9.
  • the buffer is preferably a hydrochloride buffer.
  • the hydrochloride buffer is a hydrochloride buffer containing NaCl and/or Tris-HCl.
  • the buffer for the Cas12a/crRNA digestion system may include Tris acid, sodium salt and magnesium salt.
  • the buffer contains Tris-HCl, NaCl, and MgCl 2 .
  • the buffer contains 10 mM to 50 mM Tris-HCl, 50 mM to 100 mM NaCl, and 10 mM MgCl 2 .
  • the buffer used in the Cas12a/crRNA digestion system further contains bovine serum albumin.
  • the buffer for the Cas12a/crRNA digestion system contains 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl 2 and 100 ⁇ g/mL bovine serum albumin, with a pH of 7.9.
  • the buffer used in the Cas12a/crRNA digestion system can use commercially available products, or can be configured according to known protocols.
  • the buffer used in the Cas12a/crRNA digestion system can be a commercially available product, for example, NEBuffer 3.1.
  • the Cas12a/crRNA digestion can be performed at 35°C-38°C, preferably 37°C.
  • the molar ratio of DNA to be digested with Cas12a can be 1:25 or more, for example, 1:50 or more, 1:100 or more, 1:200 or more, 1:1000 or more, 1:2000 or more, 1:3000 or more, 1:5000 or more, 1:10000 or more, or 1:20000 or more.
  • the amount of Cas12a can be appropriately increased.
  • the molar ratio of DNA to be digested to Cas12a can be 1:25 or more, preferably 1:50, 1:100 or 1: 200.
  • the molar ratio of DNA to be cleaved to Cas12a can be 1:1000 or more , For example, 1:2000 or more, 1:5000 or more, 1:10000 or more, or 1:20000.
  • the digestion is performed for 40 to 180 minutes.
  • the restriction enzyme digestion is performed for 40 min-120 min.
  • the restriction enzyme digestion is performed for 80 min-180 min.
  • Cas12a can be inactivated.
  • Cas12a is inactivated by heat treatment (for example, 65°C or higher).
  • Cas12a is inactivated by a cationic chelating agent (e.g., EDTA).
  • Cas12a is used to digest the capture carrier at 37°C for 60 minutes, wherein the buffer used in the Cas12a/crRNA digestion system is a hydrochloride buffer with a pH of 7.9; the capture carrier is combined with The molar ratio of Cas12a is 1:25; after the digestion is completed, heat treatment at 85°C for 10 min to inactivate Cas12a.
  • the buffer used in the Cas12a/crRNA digestion system is a hydrochloride buffer with a pH of 7.9
  • the molar ratio of Cas12a is 1:25; after the digestion is completed, heat treatment at 85°C for 10 min to inactivate Cas12a.
  • Cas12a is used to digest the sample containing the target DNA fragment at 37°C for 60 minutes, wherein the buffer used in the Cas12a/crRNA digestion system is a hydrochloride buffer with a pH of 7.9; The molar ratio of the sample to Cas12a is 1:200; after the digestion is completed, heat treatment at 85°C for 10 min to inactivate LbCas12a.
  • the buffer used in the Cas12a/crRNA digestion system is a hydrochloride buffer with a pH of 7.9
  • the molar ratio of the sample to Cas12a is 1:200; after the digestion is completed, heat treatment at 85°C for 10 min to inactivate LbCas12a.
  • Cas12a is used to digest the sample containing the target DNA fragment at 37°C for 120 minutes, wherein the buffer used for the Cas12a/crRNA digestion system is a hydrochloride buffer with a pH of 7.9; The molar ratio of the sample containing the target DNA fragment to Cas12a is 1:20000; after the digestion is completed, heat treatment at 85°C for 10 min to inactivate LbCas12a.
  • the buffer used for the Cas12a/crRNA digestion system is a hydrochloride buffer with a pH of 7.9
  • the molar ratio of the sample containing the target DNA fragment to Cas12a is 1:20000; after the digestion is completed, heat treatment at 85°C for 10 min to inactivate LbCas12a.
  • Cas12a is used to digest the genome at 37°C for 120 minutes, wherein the genome is embedded in low melting point agarose; the buffer used for the Cas12a/crRNA digestion system is pH 7.9 Hydrochloride buffer; the molar ratio of the genome to Cas12a is 1:20000; after the enzyme digestion is completed, heat treatment at 85° C. for 10 min to inactivate LbCas12a.
  • the ligation of the cleaved target DNA fragment and the cleaved vector with homology arms is performed by a ligase, as long as it is a ligase for sticky end ligation.
  • the ligase is selected from T4 DNA ligase and Taq ligase.
  • the target DNA fragment and the vector with homology arms are mixed and connected at a molar ratio of 1:2-2:1.
  • the target DNA fragment and the vector with homology arms are mixed at a molar ratio of 2:1-1:1 for ligation.
  • the molar ratio of the target DNA fragment and the vector with homology arms is estimated to be 1: (5-10).
  • step (3) a step of digesting the low melting point agarose embedded block is included.
  • the ligation can be performed at 4°C to 25°C, preferably 16°C; the ligation can be performed for 1 to 12 hours.
  • the ligase can be inactivated by heat treatment (for example, 65°C).
  • the host cells used in the present invention can be obtained commercially or can be prepared by methods known in the art.
  • the ligation product can be transferred into the host cell according to a scheme known in the art, and screened and verified.
  • the host cell can be selected according to needs, for example, commercialized E. coli, yeast cells, etc. can be used.
  • the electrotransformation competent cell is Escherichia coli DH10b, which is characterized in that the strain has knocked out sequence genes related to recombination, which is beneficial to maintain stable cloning.
  • the method for cloning large fragment DNA in vitro based on the CRISPR/Cas12a system provided by the present invention can efficiently realize the cloning of gene clusters.
  • the gene clusters and their ranges are new gene clusters predicted by online tools such as antiSMASH, and then these gene clusters are cloned by the method of the present invention and then expressed heterologously.
  • the present invention provides a kit for cloning large fragments of DNA by the above method
  • the kit may comprise a vector, Cas12a or a biologically active variant thereof, or an expression vector for expressing the Cas12a or a biologically active variant thereof ; And instructions for use.
  • the kit further comprises reagents for isolating the genome, Cas12a nuclease digestion buffer, crRNA in vitro transcription reagents, host cells, or any combination of the foregoing.
  • the present invention relates to in vitro large-segment DNA cloning methods, kits and related applications mediated by the CRISPR/Cas12a system. Its advantage is that it can quickly clone large-segment DNA with high GC content, and its operation is simple and does not rely on expensive equipment. , Save time and cost, and have a high positive rate and have the potential for market application.
  • (1) Construction and cutting of the capture vector prepare homology arms at both ends of the target DNA fragment, and connect the homology arms to the vector to obtain the capture vector, wherein the homology arm contains at least one A PAM site that can be recognized by Cas12a or its biologically active functional fragment or variant; based on the crRNA that is at least partially complementary bound to the homology arm, the Cas12a or its biologically active functional fragment or variant is used to capture the The carrier is cut to obtain a cut capture carrier;
  • target DNA fragments based on the crRNA and using the Cas12a or its biologically active functional fragment or variant, the sample containing the target DNA fragment is cut to obtain the target DNA fragment;
  • the cut capture vector is ligated with the target DNA fragment, and transferred/introduced into a host cell to obtain a recombinant host cell with the target DNA fragment.
  • the homology arm is at least 100 bp away from the 5'end or 3'end of the target DNA fragment, preferably at least 200 bp, more preferably at least 500 bp, and even more preferably at least 1 kb, more preferably at least 1.2 kb, more preferably at least 1.5 kb.
  • the length of the homology arm is 200bp or more, 300bp or more, 400bp or more, 500bp or more, 600bp or more, 700bp or more, 800bp or more, 900bp or more, 1000bp or more, 1100bp Above, above 1200bp, above 1300bp, above 1400bp, or above 1500bp.
  • the capture vector is selected from a plasmid, a cosmid, a bacterial artificial chromosome (BAC) or a yeast artificial chromosome (YAC).
  • step (1) before cutting the capture vector, it further comprises screening and separating the capture vector A step of.
  • the crRNA comprises a spacer sequence and a binding sequence, the spacer sequence being capable of at least partially complementary binding to the homology arm ,
  • the binding sequence can bind to the Cas12a or its biologically active functional fragment or variant.
  • step (1) the cutting is performed at a temperature of 35°C-38°C, preferably 37°C, for 40 min- 120min, preferably 50min-100min, more preferably 60min-80min.
  • step (1) the ratio between the Cas12a or its biologically active functional fragment or variant and the capture carrier
  • the ratio is 50:1 or more, preferably 80:1 or more, more preferably 100:1 or more, still more preferably 150:1 or more, and still more preferably 200:1 or more.
  • step (1) the molar ratio of the Cas12a or its biologically active functional fragment or variant to the crRNA It is 1:1 or more, preferably 2:1 or more, more preferably 4:1 or more, still more preferably 8:1 or more, and still more preferably 10:1 or more.
  • step (1) it further comprises inactivating the Cas12a or its biologically active functional fragment or variant Step; and/or a step of separating the cut capture carrier.
  • step (2) when the sample is a genome or a metagenomic group, the sample is a low melting point agarose Provided in the form of embedded blocks.
  • step (2) the cutting is performed at a temperature of 35°C-38°C, preferably 37°C, for 80 min- 180min, preferably 100min-160min, more preferably 100min-140min.
  • step (2) the Cas12a or its biologically active functional fragment or variant is compatible with the target DNA fragment
  • the molar ratio is 2,000 or more, preferably 5,000 or more, more preferably 10,000 or more, and even more preferably 20,000 or more.
  • step (2) it further comprises inactivating the Cas12a or its biologically active functional fragment or variant Step; and/or the step of separating the cut target DNA fragment.
  • step (1) and step (2) the cutting is performed at pH 7.5-8.0, preferably pH 7.9 In the buffer.
  • step (3) T4 DNA ligase and Taq ligase are used for ligation.
  • step (3) the target DNA fragment and the capture vector are in a ratio of 2:1-1:10
  • the molar ratio is mixed to make the connection.
  • step (3) for the case where the enzyme is T4 DNA ligase, the ligation is at 4°C- Perform 1-12h at a temperature of 25°C, preferably 16°C.
  • step (3) the ligation product is transferred into the host cell by electrotransformation.
  • connection product is desalted before electroconversion.
  • BGC prediction predict BGC through online tools
  • (2) Construction and cutting of the capture vector prepare the homology arms at both ends of the BGC, and connect the homology arms to the vector to obtain the capture vector, wherein the homology arms include at least one A PAM site that can be recognized by Cas12a or its biologically active functional fragment or variant; based on the crRNA at least partially complementary binding to the homology arm, the Cas12a or its biologically active functional fragment or variant is used for the The capture carrier is cut to obtain a cut capture carrier;
  • the cut capture vector is connected to the BGC, and transferred/introduced into a host cell to obtain a recombinant host cell with the BGC.
  • a method for improving the cutting efficiency of the CRISPR/Cas12a system comprising performing in a hydrochloride buffer at a temperature of 35°C-38°C, preferably 37°C, for 40min-120min, preferably 50min-100min , More preferably 60min-80min.
  • hydrochloride buffer contains sodium hydrochloride, magnesium hydrochloride and/or Tris-HCl.
  • kits for cloning large fragments of DNA in vitro comprising a vector, Cas12a or its biologically active functional fragment or variant, or expressing the Cas12a or its biologically active functional fragment or variant Expression vector; and instructions for use.
  • kit further comprises a buffer for CRISPR/Cas12a cleavage
  • the buffer is a hydrochloride buffer with a pH of 7.5-8.0, Preferably it is 7.9.
  • the crRNA used was prepared in the following way:
  • oligonucleotides as templates for crRNA transcription (5' end has a spacer sequence that is reverse complementary to the target gene fragment, the middle is the binding sequence of Cas12a protein, and the 3'end is T7 for in vitro transcription.
  • Promoter binding sequence using Taq DNA polymerase PCR buffer (Thermo Fisher Scientific) annealing; using HiScribe TM T7 rapid high-yield RNA synthesis kit (NEB) for crRNA in vitro transcription; using RNA Clean&Concentrator TM -5 kit ( Zymo Research) purified the obtained crRNA, and then quantified it using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).
  • RNase-free materials (Axygen Scientific, Union City, CA, USA) were used throughout the experiment.
  • the homology arm fragment and the vector are assembled into a capture vector by Ezmax assembly, and the molar ratio of the vector to the fragment is generally 1:2.
  • the plasmid constructed by the present invention and its preparation method involved in the examples are as follows:
  • BAC plasmid pBAC-ZL Take the BAC plasmid as the backbone, with a size of 137 kb, and an insert sequence of 128 kb in size.
  • the insert sequence exists in DDBJ/EMBL/GenBank, accession number: AEYC00000000.
  • the upstream and downstream homology arm sequences were passed through 50kb-BAC-arm-up-F and 50kb-BAC-arm-up-R( SEQ ID NO: 1-SEQ ID NO: 2, the sequence of the upstream homology arm 50kb-BAC-arm-up is SEQ ID NO: 3, 990bp) and BAC-arm-dn-F and BAC-arm-dn-R (SEQ ID NO: 4-SEQ ID NO: 5, the sequence of the downstream homology arm 50kb-BAC-arm-dn is SEQ ID NO: 6,228 bp), there is a LacZ selection marker (its The nucleic acid sequence is shown in SEQ ID NO: 7, 620 bp, and the upstream and downstream primers are BAC-lacZ-F (SEQ ID NO: 8) and BAC-lacZ-R (SEQ ID NO:
  • the upstream and downstream homology arm sequences were passed through 80kb-BAC-arm-up-F and 80kb-BAC-arm-up-R( SEQ ID NO: 10-SEQ ID NO: 11, the sequence of the upstream homology arm 80kb-BAC-arm-up is SEQ ID NO: 12, 880bp) and BAC-arm-dn-F and BAC-arm-dn-R (SEQ ID NO: 4-SEQ ID NO: 5, the sequence of the downstream homology arm 80kb-BAC-arm-dn is SEQ ID NO: 6,228 bp), there is a LacZ selection marker (its The nucleic acid sequence is shown in SEQ ID NO: 7, 620 bp).
  • Buffer is an important factor that affects the efficiency of digestion. Under the premise that other conditions remain unchanged, the pH and ionic strength of different buffer solutions are different, and the cutting efficiency of Cas12a is significantly different.
  • the Cas12a protein is prepared as follows: the LbCas12a protein is overexpressed in pET28a, and then purified by fast protein liquid chromatography (FPLC; AKTA Explorer 100, GE Healthcare) using methods such as Liang, Mindong, etc., "A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules.”Nature communications vol.10,1 3672.14 Aug.2019, doi:10.1038/s41467-019-11648-1.
  • FPLC fast protein liquid chromatography
  • the DNA fragment for Cas12a cleavage was prepared by using the capture plasmid pBAC2015-C50 as a template, forward primer (50kb-BAC-arm-up-F, SEQ ID No: 1) and reverse primer (BAC-lacZ- R, SEQ ID NO: 9)) was used as a primer to perform PCR to obtain a test fragment 1 (ie, upstream homology arm + lacZ) with a length of 1610 bp.
  • forward primer 50kb-BAC-arm-up-F, SEQ ID No: 1
  • BAC-lacZ- R reverse primer
  • crRNA 50kb-BAC-up-crRNA, also called test fragment 1-crRNA
  • SEQ ID No: 13 The sequence of crRNA (50kb-BAC-up-crRNA, also called test fragment 1-crRNA) used for test fragment 1 cleavage is shown in SEQ ID No: 13.
  • B7204S Use different buffer cutsmart buffer (B7204S), NEBuffer2.1 (B7202S), NEBuffer3.1 (B7203S) and NEBuffer4 (B7004S) purchased from New England Biolabs (NEB).
  • NEBuffer 2.1 and NEBuffer 3.1 are hydrochloric acid buffers, and the difference between the two lies in the salt ion concentration.
  • Medium salt buffer NEBuffer 2.1 (pH 7.9) components are 50mM sodium chloride, 10mM Tris-hydrochloric acid, 10mM magnesium chloride, 100 ⁇ g/mL bovine serum albumin;
  • high salt buffer NEBuffer 3.1 (pH 7.9) components are 100mM sodium chloride, 50mM Tris-hydrochloric acid, 10mM magnesium chloride, 100 ⁇ g/mL bovine serum albumin.
  • NEBuffer 4 and CutSmart Buffer are both acetate buffers.
  • NEBuffer 4 (pH 7.9) consists of 50 mM potassium acetate, 20 mM Tris-acetic acid, 10 mM magnesium acetate, and 1 mM dithiothreitol. CutSmart Buffer is an upgraded version of NEBuffer 4. It contains dithiothreitol and bovine serum albumin.
  • the enzyme digestion buffer add the digestion system.
  • the digestion system is shown in the following table, and each digestion system is reacted at 37°C for 1 hour.
  • the enzyme digestion results were analyzed by nucleic acid electrophoresis. Among them, the test fragment 1 that was not digested was used as a control. The results are shown in Figure 2. Among them, Casl2a has higher cutting activity in hydrochloric acid buffers NEBuffer 2.1 and NEBuffer 3.1, Cas12a The cutting efficiency in NEBuffer 3.1 is the best, and the target fragment is almost completely cut. However, NEBuffer 4 and CutSmart Buffer have poor activity, and there are obviously uncut target fragments in the gel image. It can be seen that for Cas12a cleavage, it has better activity in high-salt hydrochloride buffer.
  • reaction time in the digestion system is one of the important factors affecting the cutting efficiency. Without changing other conditions, the longer the reaction time, the higher the cleavage efficiency of Cas12a; however, as the reaction time continues to increase, it may cause non-specific cleavage of Cas12a.
  • Use test fragment 1 at the same time, use the same Cas12a protein and crRNA as 1-1 for digestion reaction.
  • the digestion system is shown in the following table. The digestion system was reacted at 37°C for 20 min, 40 min, 60 min, and 80 min, respectively.
  • the results of restriction digestion were analyzed by nucleic acid electrophoresis, and the 1610 bp test fragment 1 that was not digested was used as a control.
  • the results are shown in FIG. 3.
  • the results in Figure 3 show that when the cutting time is less than 60 minutes, the electrophoresis results show that there are obviously uncut target PCR fragments.
  • the cutting time was extended to 60 minutes and 80 minutes, Cas12a almost completely cut the target PCR fragments, and there was no significant difference in cutting efficiency at 60 minutes and 80 minutes.
  • the enzymolysis time is not directly proportional to the enzymolysis efficiency.
  • the target fragment will also be degraded if the reaction time is too long.
  • the above-mentioned target fragment was digested overnight (more than 12 hours), and the electrophoresis result was diffuse and there was no target band.
  • Test fragment 1 as a template for restriction digestion
  • test fragment 1 and crRNA were used for the digestion reaction.
  • the digestion system is shown in Table 2. Wherein, the molar ratio of test fragment 1 to Cas12a protein is 1:12.5, 1:25, 1:50, 1:100, and 1:200.
  • the digestion system was carried out at 37°C for 60 minutes.
  • Control is a template PCR fragment.
  • the template-protein molar ratio is 1:25-1:100, there is no significant difference in the cutting effect, and the template DNA is almost completely cut.
  • the template-protein molar ratio is 1:12.5, that is, when the amount of Casl2a and crRNA is small, a large amount of template DNA is not cut.
  • the Cas12a protein which is the same as 1-1
  • the crRNA 50kb-BAC-up-crRNA, SEQ ID No: 13
  • the crRNA of the source arm (BAC-dn-crRNA, SEQ ID No: 14), using the capture plasmid pBAC2015-C50 as a template to continue the cutting experiment.
  • the reaction system is shown in Table 3 below.
  • the Cas12a cleavage system for PCR fragments or capture plasmids can be performed in Table 3 below.
  • the principle of CAT-FISHING is to connect the sticky ends of two linear DNA fragments by DNA ligase.
  • the sticky ends are produced by CRISPR/Cas12a cleavage guided by paired crRNAs. Therefore, this study first used the small fragment capture experiment of Amp R (ampicillin resistance gene) to compare the application of commercial NEB restriction endonuclease and CRSIPR/Cas12a enzymatic digestion of two different cohesive ends in the clone assembly. The specific process is shown in Figure 5. This step verified the feasibility of the CAT-FISHING gene manipulation platform, and completed the evaluation of the CAT-FISHING cloning efficiency.
  • the pGY2020 (pCC2-FOS plasmid (purchased from EpicentreBio) with the Ch1 resistance gene) and pUC19 plasmid (purchased from NEB) were used. Refer to Figure 5 for specific operations.
  • the crRNA used is 50kb-BAC-up-crRNA and BAC-dn-crRNA.
  • the capture plasmid pGY2020 assembled through the pCC2-FOS plasmid backbone contains two PAM sites (PAM1 and PAM2) and two NEB restriction endonuclease sites (EcoR I and Hind III).
  • PAM1 and PAM2 two PAM sites
  • EcoR I and Hind III two NEB restriction endonuclease sites
  • Cas12a mediated by 18nt crRNA usually cuts the 14th position of the non-complementary strand, resulting in a sticky end with a length of 8nt, but sometimes the position of the cut point will swing, resulting in the deletion of bases at the junction.
  • CAT-FISHING method we randomly selected three PCR positive clones for gene sequencing. Specific data are shown in Table 4.
  • the crRNA used in the experiment was prepared by in vitro transcription from DNA primers.
  • the crRNA primer template consists of three parts, the 5'end 18bp and the target gene fragment reverse complementary spacer sequence, the middle 21bp is the binding sequence of the Cas12a protein, and the 3'end is the binding sequence of the T7 promoter during in vitro transcription.
  • the selection of 5'spacer sequence is the key to crRNA design.
  • Figure 8 is the optimization result of three 50kb-BAC-up-crRNA.
  • This experiment compares the double-enzyme digestion effect of Cas12a on the upstream homology arm of 50kb-BAC under the mediation of three pairs of different crRNAs, using the capture plasmid pBAC2015-C50 as a template.
  • four different sets of Cas12a/crRNA:DNA molar ratios were set.
  • the experiment used the same downstream crRNA (BAC-dn-crRNA, the sequence is SEQ ID NO: 14).
  • Control is a PCR sample of 50kb-BAC upstream homology arm-lacZ-downstream homology arm.
  • the third pair of crRNA has a better cutting effect, the two target bands are clear, and the residual amount of template is less; when the first pair of crRNA is used, the DNA template is cut into several fragments of different sizes by Cas12a.
  • the first upstream crRNA has poor specificity.
  • the second pair of crRNA is not non-specific, when using the same template-protein molar ratio, the efficiency is significantly lower than that of the first pair of crRNA-mediated cleavage. There is only one clearly visible band of interest, and most templates are not. Get fully cut. Therefore, the third pair of crRNA was selected for subsequent experiments (50kb-BAC-up-crRNA and BAC-dn-crRNA).
  • a BAC plasmid special kit was used to prepare a high-purity pBAC-ZL template.
  • the cleavage time of 137kb BAC plasmid in vitro and the molar ratio of Casl2a/crRNA/DNA were further optimized.
  • the in vitro cleavage time and template-protein molar ratio were determined to be 120min and 1:2000, respectively.
  • the cutting system is shown in Table 6. Under this cutting condition, the pBAC-ZL plasmid digested with CRISPR/Cas12a is subjected to pulsed field gel electrophoresis (PFGE) and an obvious band of interest can be observed.
  • PFGE pulsed field gel electrophoresis
  • pBAC-ZL was cleaved by Cas12a into two fragments of 87kb and 49kb under the guidance of 50kb-BAC-up/dn-crRNA.
  • 80kb-BAC-up/dn-crRNA-mediated Cas12a cuts pBAC-ZL into two fragments, 57kb and 79kb.
  • the cleavage of the capture plasmid was the same as in Example 1.
  • the digested products of the above-mentioned capture plasmid pBAC2015-C50 or pBAC2015-C80 and the target fragment are subjected to alcohol precipitation and recovery.
  • T4 DNA ligase to ligate the linearized capture vector and the target fragment.
  • the large fragment clone is introduced into the recipient cell by electrotransformation. Since the electrotransformed sample cannot contain too much salt ions, the connection product should be transferred to the desalting gel after the reaction is completed. In the process, desalination treatment is performed, and then all the desalination products are introduced into DH10b.
  • the CHEF-DR III instrument (Bio-Rad, Richmond, CA) can be used to analyze larger DNA fragments by pulsed field gel electrophoresis (PFGE).
  • PFGE pulsed field gel electrophoresis
  • the electrophoresis conditions were in 0.5 ⁇ TBE buffer, 0.5V agarose in 6V/cm PFGE for a switching pulse time of 1-25 seconds, lasting 16-18h.
  • the ligation system using T4 DNA ligase is as follows:
  • Desalting transfer the connected sample to 0.1M glucose/1% agarose gel, and desalting on ice for 1 to 2 hours, the sample can be used for electroconversion.
  • Electroporation Using Bio-Rad GenePulser XcellTM system in a 2mm electroporation cup (electroporation conditions: 2500V, 200 ⁇ and 25 ⁇ F). Then, add 1 mL of SOC medium (tryptophan 20g/L, yeast extract 5g/L, NaCl 0.5g/L, KCl 2.5mM, MgCl 2 10mM, glucose 20mM) to the E. coli cells in the electrorotor, and then The mixture was transferred to a 15 mL Falcom TM tube. After shaking at 200 rpm at 37°C for 1 hour, the strains were collected and spread on selective (containing chloramphenicol resistance and X ⁇ gal) LB agar plates. The plates were incubated overnight at 37°C, and then transformants were screened and verified by PCR using the following primers.
  • SOC medium tryptophan 20g/L, yeast extract 5g/L, NaCl 0.5g/L, KCl 2.5mM, M
  • the verification primers are designed in the upstream, midstream and downstream of the target fragment respectively.
  • the specific data are shown in Table 7 and Figure 13.
  • cloning vectors pBAC2015-50kb-BAC and pBAC2015-80kb-BAC containing 50kb and 80kb fragments were obtained, respectively.
  • the CAT-FISHING technology for the cloning of 50kb DNA fragments has a positive rate of about 95% among dozens to hundreds of transformants.
  • the capture efficiency of 80kb DNA fragments dropped significantly, and the number of transformants and the positive rate were lower than those of 50kb ( Figure 13).
  • the CAT-FISHING technology can efficiently clone 50kb and 80kb large fragment gene clusters from the 137kb pBAC-ZL plasmid. It should be noted that for 80kb DNA fragments, cloning is significantly more difficult.
  • the size and sequence complexity of the bacterial genome is much larger than that of the BAC plasmid.
  • the genome (GenBank Assembly: GCA_000359525.1) of the donor strain S. albus J1074 used in this experiment is 6.8Mb, and the GC content is as high as 73%.
  • the template 137kb BAC is a high-purity DNA extracted by the kit.
  • the genomic template is prepared by DNA embedding, and its purity is much lower than that of the BAC plasmid template. Combining the above two factors, we optimized the conditions of in vitro restriction digestion and the method of genome embedding.
  • Streptomyces genome embedding The specific steps of Streptomyces genome embedding are as follows:
  • the capture vector is based on pBAC2015, and is composed of primers for the 49kb fragment (50kb-up-hom-arm-F (SEQ ID No: 34) and 50kb-up-hom-arm-R (SEQ ID No: 35) and 50kb-dn-hom-arm-F (SEQ ID No: 36) and 50kb-dn-hom-arm-R (SEQ ID No: 37), the upstream homology arm 49kb-up-hom-arm (SEQ ID No: 38) and the downstream homology arm 49kb-dn-hom-arm (SEQ ID No: 39) are 727bp and 611bp, respectively) and the primer for the 87kb fragment ((80kb-up-hom-arm-F(SEQ ID No: 40) and 80kb-up-hom-arm-R (SEQ ID No: 41) and 80kb-dn-hom-arm-F (SEQ ID No: 42) and 80kb-dn-hom-arm-R (SEQ
  • the first step is the preparation of the embedded block. This step takes 4 to 5 hours. Prepare the genomic embedding block according to the optimized method, wash the embedding block for cutting with ultrapure water twice, and soak it in 100 ⁇ L 1 ⁇ NEBuffer 3.1 for 30 min. The remaining embedded blocks were placed in ethanol at 75°C and stored frozen at -20°C.
  • the second step is the enzymatic hydrolysis of the genome. This step takes 2 hours. According to the optimized cutting conditions, use the corresponding crRNA to mediate CRISPR/Cas12a to specifically cut the embedded block and the capture plasmid.
  • the target band can be observed after PFGE.
  • 50kb-up/dn-crRNA-mediated Cas12a cuts the embedding block to produce a 49kb band of interest
  • 80kb-up/dn-crRNA mediated Cas12a cuts the embedding block to produce a 87kb band.
  • agarase enzyme digests the cut product, which takes 30 minutes. Transfer the genomic embedding block after the digestion treatment in step 2 to an appropriate amount of agarase solution, and digest it at 42°C for 30 minutes to completely dissolve the embedding block to obtain the target gene cluster mixture.
  • the fourth step, the ligation reaction takes 3 to 4 hours.
  • the target gene cluster mixture does not need to be separated and purified, and can be directly used in enzyme linkage experiments. Take 500ng each of the purified linearized capture plasmid and target gene cluster, use T4 DNA ligase for ligation, ligate the system 30 ⁇ L, and react for 1 to 2 hours.
  • the fifth step, desalination of the ligation product took 2h. Transfer all the connected products to the configured desalting gum and let stand at 4°C for 2h.
  • the sixth step, electric conversion takes 2h. Transfer all the desalted products (about 30 ⁇ L) to 170 ⁇ L high-concentration E.
  • coli DH10b electroporation competent state let stand on ice for 10min, transfer to a 2mm electroporation cup, and perform electroporation under the conditions of voltage 2500V, capacitance 25 ⁇ F, and resistance 200 ⁇ , Quickly add 500 ⁇ L of SOC medium, incubate at 37°C for 1h, transfer all 700 ⁇ L of bacterial solution to LB plate containing X ⁇ gal and chloramphenicol resistance, spread evenly, blow-dry it on a clean bench, and incubate at 37°C for 16 ⁇ The single clone can be seen at 20h.
  • the competent efficiencies used for electroporation were all 0.8 ⁇ 1 ⁇ 10 8 cfu/ ⁇ L.
  • cloning vectors pBAC2015-49kb-J1074, pBAC2015-87kb-J1074 and pBAC2015-139kb-J1074 containing 49kb paulomycin gene cluster, 87kb surugamides gene cluster, and 139kb candidadin gene cluster were obtained.
  • Table 10 The cloning efficiency of targeted cloning of high GC gene clusters from genomic DNA.
  • ElectroMAX TM DH10B TM cell (Thermo Fisher Scientific, Invitrogen TM ) can increase the number of transformants.
  • the Streptomyces conjugative transfer element, the replicon and the selection marker aac(3)IV-oriT-attP(+C31)-int(+C31) were integrated into the recombinant plasmid by Rec/ET recombination technology.
  • the target plasmid pBAC2015-87kb-J1074-int in DH10b was integrated into the chromosome of S.albus Del14. Verify the binder and scale-up fermentation in R5A medium.

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Abstract

L'invention concerne un procédé de clonage in vitro de grands fragments d'ADN à teneur en GC élevée médié par un système CRISPR/Cas12a, un kit de réactifs et des utilisations associées. Le procédé de clonage utilise Cas12a/crRBA pour un clivage spécifique de chaque extrémité d'un ADN cible pour acquérir une séquence d'ADN cible, construit un vecteur de clonage par clonage des séquences d'ADN à chaque extrémité de l'ADN cible, utilise Cas12a/crARN pour cliver le vecteur de clonage pour acquérir un vecteur de linéarisation et relie, par l'intermédiaire d'une ADN ligase, des extrémités collantes formées lors du clivage, mettant ainsi en œuvre le clonage de l'ADN cible.
PCT/CN2020/120332 2020-06-22 2020-10-12 Procédé de clonage in vitro de grands fragments d'adn à base de crispr/cas12a et applications associées WO2021258580A1 (fr)

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