CN106119269B

CN106119269B - Method for preparing linear single-stranded DNA in escherichia coli

Info

Publication number: CN106119269B
Application number: CN201610464596.XA
Authority: CN
Inventors: 张平静; 冯文建; 杨扬; 王晋康; 朱远源
Original assignee: Biomics Biotechnologies Co Ltd
Current assignee: Biomics Biotechnologies Co Ltd
Priority date: 2016-06-23
Filing date: 2016-06-23
Publication date: 2019-12-06
Anticipated expiration: 2036-06-23
Also published as: CN106119269A

Abstract

The invention discloses a method for preparing linear single-stranded DNA, which comprises the following steps: constructing a Cas expression element capable of expressing the Cas nickase and integrating the Cas expression element into a plasmid; constructing a sgRNA gene transcription expression element compatible with the Cas expression element in an escherichia coli cell, and constructing a plasmid to obtain a sgRNA expression plasmid; the Cas nickase expression plasmid and the sgRNA expression plasmid are jointly transformed into an escherichia coli competent cell; culturing escherichia coli to logarithmic growth phase, inducing Cas nickase gene expression and suicide sgRNA expression, and allowing the Cas nickase to cooperate with the sgRNA to self-shear double-stranded DNA of sgRNA expression plasmid and form single-stranded break to generate linear single-stranded DNA; extracting linear single-stranded DNA.

Description

method for preparing linear single-stranded DNA in escherichia coli

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a method for preparing linear single-stranded DNA (LssDNA) in escherichia coli and a kit for implementing the method.

Background

Single-stranded DNA (ssDNA) has wide applications in the biological field, such as DNA sequencing, site-directed mutagenesis, probe preparation, aptamer screening, plasmid microcarrier preparation for gene therapy, DNA origami, and nano-delivery drug delivery. At present, the large-scale preparation of long-chain ssDNA with more than 200nt is always a problem to be solved in the field.

To date, there are a wide variety of methods for preparing single-stranded ssDNA in vitro, such as chemical synthesis; asymmetric PCR; separating the avidin-labeled single chains by using streptavidin particles; digesting the phosphorylated single strand by lambda exonuclease; performing rolling circle PCR amplification; denaturation after plasmid cleavage by restriction enzymes, etc. These methods also have some disadvantages, such as: 1. the length of ssDNA prepared is limited, usually less than 200 nt; 2. requires cleavage by means of biological enzymes in an in vitro reaction and is therefore costly; 3. the process can not be enlarged for large-scale production and is only limited to laboratory scale.

In addition to the above methods, one skilled in the art can also scale-up ssDNA using bioreactors, e.g., phage/bacteria, virus/cell systems. The M13 phage is able to infect an E.coli host and secrete the phage particles from the infected cells, while the host cells continue to grow and divide. The genome of this phage is a circular single-stranded DNA molecule, 6407 nucleotides in length, containing the genetic information required for DNA replication and phage propagation, with which long-chain ssDNA can be produced in large quantities. However, the length of the exogenous DNA fragment which can be accommodated by the circular single-stranded DNA is very limited, about 300 nt and 400nt, and sometimes the exogenous DNA fragment is easily lost; in addition, the circular ssDNA prepared by the method contains a large number of phage genome sequences. The modified phagemid (phagemid) can partially solve the problems and improve the length of the exogenous DNA, but has the limitation. For example, 1. helper phage infection is required to achieve production of replicated ssDNA; 2. sometimes the yield of single-stranded DNA is low and reproducibility is poor; 3. the phage production mode has certain special requirements on equipment and experimental environment; 4. the ssDNA prepared by this method is circular and contains a large number of phage genome sequences.

Eukaryotic/viral systems can also produce ssDNA, and AAV viral particles containing the ssDNA genome can be packaged by co-transfecting eukaryotic cells, including mammalian cells, insect cells, yeast cells, and the like. However, this method has a limited productivity and there are many concerns about biological safety. In addition, the production process involves cell culture and other processes, which results in high preparation cost.

The CRISPR/Cas system plays an important role in prokaryotic antiviral defense, is present in 90% and 40% of archaea and bacterial genomes, respectively, and can be used to combat invading viruses and foreign DNA. CRISPR/Cas systems are largely divided into three categories: form I, form II and form III. Where type II requires a protein called Cas9 for DNA interference. In recent years, the type II CRISPR/Cas system is used in higher eukaryotes to achieve genome editing at the cellular and organism level, and the global application of the CRISPR-Cas9 technology has increased explosively over the past few years. The working principle of this system is that crRNA (CRISPR-derived RNA) is combined with tracrRNA (trans-activating RNA) by base pairing to form a tracrRNA/crRNA complex, and the nuclease Cas9 protein is guided to cleave double-stranded DNA at the sequence target site paired with the crRNA. The sgrna (single guide rna) of the artificially-modified tracrRNA/crRNA also has a guiding function, and is enough to guide Cas9nickase to perform site-directed cleavage on DNA containing a PAM sequence, so that a gene editing function is realized. With the clear CRISPR/Cas9 mechanism, the technology is widely applied to genome editing of different species, and the gene editing system is efficient, simple and easy to operate. Wild-type Cas9 has multiple domains, creating a DNA double strand break through the two nuclease domains RuvC and HNH, and researchers often convert one of the two key residues in Cas9 nuclease domain to alanine (D10A or H840A) during eukaryotic gene editing to reduce off-target effects, forming Cas9nickase (Cas9-nickase, Cas9 n). Cas9n requires the assistance of two sgrnas to generate a DNA double strand break, greatly enhancing recognition target sequence specificity compared to wild-type spCas 9. D10A (i.e., aspartic acid D at position 10 mutated to alanine a) mutant Cas9n cleaves only sgRNA complementary strand DNA, while H840A (i.e., histidine H at position 840 mutated to alanine a) mutant Cas9n cleaves only sgRNA non-complementary strand DNA. Cas9, in which both the D10A and H840A mutations were present, was designated dCas9, which still retained DNA binding activity but did not have any enzymatic cleavage activity.

Disclosure of Invention

In order to overcome the defect of large-scale production of linear single-stranded DNA (particularly linear long single-stranded DNA (LssDNA)) of more than 200nt in the prior art, the invention constructs a CRISPR/Cas 9nickase suicide system by utilizing a genetic engineering technology according to the principle of a CRISPR/Cas system, and implements large-scale preparation of the LssDNA by taking escherichia coli which is most commonly used in the field of biotechnology as a production bacterium.

Accordingly, a first object of the present invention is to provide a method for preparing linear single-stranded DNA in Escherichia coli.

it is a second object of the invention to provide a kit for carrying out the method.

In order to achieve the purpose, the invention adopts the following technical scheme:

A method for preparing linear single-stranded DNA in Escherichia coli, comprising the steps of:

A) Constructing a Cas expression element capable of expressing Cas nickase, wherein the Cas expression element comprises an operator, a promoter, a Cas nickase gene sequence, a terminator, a replicon and a screening marker gene, and the Cas nickase is spCas9 derived from streptococcus pyogenes or sacAS9 derived from staphylococcus aureus, preferably spCas9 derived from streptococcus pyogenes;

B) Integrating the Cas expression element obtained in the step A) into a plasmid or genome to obtain a Cas nickase expression plasmid or a recombinant genome;

C) Constructing a sgRNA gene transcription expression element compatible with the Cas expression element in an Escherichia coli cell, wherein the sgRNA gene transcription expression element comprises an operator, a promoter, a sgRNA gene sequence, a terminator, a replicon and a screening marker gene,

D) Constructing the sgRNA gene transcription expression element obtained in the step C) into a plasmid to obtain a sgRNA expression plasmid, wherein the plasmid contains a PAM sequence serving as a suicide sgRNA target and a sequence corresponding to the linear single-stranded DNA;

E) Co-transforming the Cas nickase expression plasmid obtained in the step B) and the sgRNA expression plasmid obtained in the step D) into an Escherichia coli competent cell, or transforming the sgRNA expression plasmid obtained in the step D) into an Escherichia coli competent cell containing the recombinant genome;

F) Culturing escherichia coli to logarithmic growth phase, inducing Cas nickase gene expression and suicide sgRNA transcription expression, allowing the Cas nickase to cooperate with the sgRNA to self-shear double-stranded DNA of sgRNA expression plasmid and form single-stranded break, and generating linear single-stranded DNA through the cooperation of a theta replication mechanism of the plasmid;

G) Extracting linear single-stranded DNA in a DNA product self-sheared by the sgRNA gene transcription expression element.

Preferably, the above-mentioned Cas nickase is Cas9(D10A) shown in SEQ ID No:1 or Cas9(H840A) shown in SEQ ID No: 2.

Preferably, the PAM sequence is a5 '-Nx-NGG-3' sequence, wherein N represents any one of bases A, T, C, G, and x represents any natural number between 10 and 30. Nx in a sequence does not mean a repeated sequence of a certain base such as AA … … AA, TT … … TT, CC … … CC, GG … … GG, but means only x in the number selected from four bases A, T, C and G, as can be understood by those skilled in the art.

Preferably, the operons in the Cas expression element and the sgRNA gene transcription expression element described above are each independently selected from the group consisting of: tetracycline operon, lactose operon, galactose operon, arabinose operon, rhamnose regulator, pR/pL temperature control operon.

Preferably, the operator in the Cas expression element described above is a tetracycline operator (Tet-on).

Preferably, the selection marker genes in the Cas expression element and the sgRNA gene transcription expression element described above are each independently selected from the group consisting of: chloramphenicol resistance gene, ampicillin resistance gene, kanamycin resistance gene.

Preferably, the promoter in the sgRNA gene transcription expression element is selected from the group consisting of: TacI, LacZ, LacI, J23100.

Preferably, the replicon in the Cas expression element described above is selected from p15 and pSC 101.

Preferably, the replicon in the sgRNA gene transcription expression element described above is selected from pMB1 and ColE 1.

Preferably, the Cas nickase expression plasmid described above is selected from the group consisting of: pETetCas9-Nickase shown in SEQ ID No:3, pETetCas9-Nickase (pSC101) shown in SEQ ID No:4, and pPrPl Cas9Nickase shown in SEQ ID No: 5.

In the SEQ ID No. 3 of the sequence of pETetCas9-Nickase, the sequence at positions 7-704 is a tetracycline operator; the sequence at position 740-4846 is Cas9nickase DNA; the sequence at positions 5146-5691 is the p15 replicon.

In the sequence SEQ ID No:4 of pETetCas9-Nickase (pSC101), the sequence at positions 7-704 is the tetracycline operator; the sequence at position 740-4846 is Cas9nickase DNA; the sequence at position 5253-6473 is a pSC101 replicon.

In the sequence SEQ ID No. 5 of pPrPL Cas9Nickase, the sequence at position 198-1323 is pR/pL temperature control operon; the sequence at position 1373-5479 is Cas9nickase DNA; the sequence at position 5779-6324 is the p15 replicon.

Preferably, the sgRNA expression plasmid described above is selected from the group consisting of: pJ23sgRNA-EGFP shown in SEQ ID No. 6 and pJ23sgRNA-GFPuv (TacI) shown in SEQ ID No. 7.

In the sequence SEQ ID No. 6 of pJ23sgRNA-EGFP, the 431-494 th sequence is the J23100 promoter; the sequence 1896-2444 is the ColE1/pMB1 replicon.

In the sequence SEQ ID No. 7 of pJ23sgRNA-GFPuv (TacI), the sequence at position 428-481 is the TacI-LacI promoter; the sequence at position 761-824 is the J23100 promoter; the sequence 2267-2855 was the ColE1/pMB1 replicon.

A kit for preparing linear single-stranded DNA, comprising: a Cas nickase expression plasmid comprising a Cas expression element that can express a Cas nickase; a sgRNA expression plasmid comprising a sgRNA gene transcription expression element compatible with the Cas expression element described above in an escherichia coli cell; and host E.coli; the Cas Nickase expression plasmid is selected from pETetCas9-Nickase shown in SEQ ID No. 3, pETetCas9-Nickase (pSC101) shown in SEQ ID No. 4 and pPrPl Cas9Nickase shown in SEQ ID No. 5; the sgRNA expression plasmid is selected from pJ23sgRNA-EGFP shown in SEQ ID No. 6 and pJ23sgRNA-GFPuv (TacI) shown in SEQ ID No. 7.

Preferably, the kit can also be used for screening the cleavage efficiency of the Cas9/sgRNA complex on the target DNA.

By the method provided by the invention, the suicide sgRNA with the optimal shearing efficiency can be screened by using a CRISPR/Cas nickase suicide system in escherichia coli, and linear long single-stranded DNA (LssDNA) is produced. In the suicide system, the Cas nickase can cut plasmid double-stranded DNA in cooperation with the suicide sgRNA to generate single-stranded break, and finally a large amount of self-cleavage product LssDNA is generated. The method can realize the large-scale production of linear single-stranded DNA, especially linear long single-stranded DNA of more than 200 nt.

Drawings

Fig. 1 is a schematic diagram of the CRISPR/Cas nickase suicide system of the present invention for producing linear long single-stranded DNA.

Fig. 2 is a structural schematic diagram of a gene transcription expression element of the CRISPR/Cas suicide system in example 1. Wherein figure 2A is a Cas nickase expression element; fig. 2B is an sgRNA gene transcription expression element.

fig. 3 is a photograph of a petri dish of e.coli transformed with different types of Cas9 expression plasmids (Cas9n, wtCas9, dCas9) in example 2, showing the effect of the CRISPR/Cas nickase suicide system on bacterial growth.

Fig. 4 is an agarose gel electrophoresis of the products of the different Cas9 expression plasmids (Cas9n, wtCas9, dCas9) transformed into e.coli in example 2, showing the effect of the CRISPR/Cas nickase suicide system on plasmid replication.

Fig. 5 is an agarose gel electrophoresis of products after co-transforming 491, 911, 107, 140 and 385 five target sgRNA expression plasmids and different types of Cas9 expression plasmids (Cas9n and dCas9) in example 3 into escherichia coli, and shows the result of screening the "moderate efficiency" sgRNA target by the CRISPR/Cas nickase suicide system. In the figure, each strip: 1. 2: 49D; 3. 4: 49N 1; 5. 6: 49N 2; 7. 8: 49N 3; 9. 10: 49N 4; 11. 12: 91D; 13. 14:91N 1; 15. 16: 91N 2; 17. 18: 107N 1; 19. 20: 140N 1; 21. 22: 385N 1. Wherein 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 are non-inducible groups, and 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 are tetracycline inducible groups.

fig. 6 is an agarose gel electrophoresis of products after the sgRNA expression plasmids of target spots 912, 913, 914, 492, 493 and 494 and the Cas expression plasmid are cotransformed into escherichia coli in example 4, and shows the results of adjusting the length of the sgRNA target spot to screen for "moderate shearing efficiency" sgRNA. In the figure, each strip: 1. 2: 911N; 3. 4: 912N; 5. 6: 913N; 7. 8: 914N; 9. 10: 491N; 11. 12: 492N; 13. 14: 493N; 15. 16: 494N. All samples were tetracycline-induced plasmids extracted after self-shearing.

FIG. 7 is an agarose gel electrophoresis of products obtained after co-transforming Escherichia coli with 491-. In the figure, each strip: 1: 492D; 2. 3: 492N; 4. 5: 913N; 6: OriR 1D; 7. 8: oriR 1N. All samples were tetracycline-induced plasmids extracted after self-shearing. D represents that the suicide sgRNA and dCas9 expression plasmid co-transform Escherichia coli; n represents suicide sgRNA co-transformed with Cas9N expression plasmid into e.

Fig. 8 is an agarose gel electrophoresis of products of escherichia coli cotransformed with the sgRNA expression plasmids of target sites 492, 913 and Ori1R and the Cas expression plasmid in example 5, and shows the self-cleavage effect of the CRISPR/Cas nickase suicide system on different sgRNA expression plasmids. In the figure, each strip: 1: 492N; 2: 913N; 4-6: oriR 1N. All samples were tetracycline-induced plasmids extracted after self-shearing. D represents that the suicide sgRNA and dCas9 expression plasmid co-transform Escherichia coli; n represents suicide sgRNA co-transformed with Cas9N expression plasmid into e.

Fig. 9 is an agarose gel electrophoresis image (SYBR Green I staining) of products after co-transformation of 491, 492, 493, 913 and 914 target sgRNA expression plasmids and Cas expression plasmids into escherichia coli in example 5, and shows the self-cleavage effect of the CRISPR/Cas nickase suicide system on different sgRNA expression plasmids. In the figure, each strip: 1: 491D; 2: 491N; 3: 492N; 4: 493N; 5: 913N; 6: 914N. All samples were tetracycline-induced plasmids extracted after self-shearing. D represents that the suicide sgRNA and dCas9 expression plasmid co-transform Escherichia coli; n represents suicide sgRNA co-transformed with Cas9N expression plasmid into e.

Fig. 10 is agarose gel electrophoresis (acridine orange staining) of products of co-transformation of 491,493 target sgRNA expression plasmids and Cas expression plasmids into escherichia coli in example 5, showing self-cleavage effect of CRISPR/Cas nickase suicide system on different sgRNA expression plasmids. In the figure, each strip: 1: 491D; 2: 491N; 3: 913N. All samples were tetracycline-induced plasmids extracted after self-shearing. D represents that the suicide sgRNA and dCas9 expression plasmid co-transform Escherichia coli; n represents suicide sgRNA co-transformed with Cas9N expression plasmid into e.

FIG. 11A is an agarose gel electrophoresis of restriction endonuclease digested self-sheared DNA products of example 5. In the figure, each strip: 1: 491D; 2: 491N; 3: 913N; 4. 913D; 5. 491N; 6. 913N; m, marker, wherein 1-3 are EcoRI digests and 4-6 are PstI digests.

FIG. 11B is an agarose gel electrophoresis of restriction endonuclease digested self-sheared DNA products of example 5. In the figure, each strip: 1: 491D; 2: 491N; 3: 913D; 4. 913N; 5. 491N; 6. 913N; m: marker, 1-4 HpaII digestion products, 5 and 6 undigested control plasmids. All samples were tetracycline-induced plasmids extracted after self-shearing. D represents that the suicide sgRNA and dCas9 expression plasmid co-transform Escherichia coli; n represents suicide sgRNA co-transformed with Cas9N expression plasmid into e.

FIG. 12 is an agarose gel electrophoresis image of the digested self-sheared DNA product of nuclease S1 in example 5. In the figure, each strip: 1-2: 491D; 3-4: 491N; 5-6: 913N; m: marker, wherein 1, 3 and 5 are products digested by nuclease S1, and 2, 4 and 6 are undigested control plasmids. All samples were tetracycline-induced plasmids extracted after self-shearing. D represents that the suicide sgRNA and dCas9 expression plasmid co-transform Escherichia coli; n represents suicide sgRNA co-transformed with Cas9N expression plasmid into e.

FIG. 13 is a drawing showing the agarose gel recovered from sheared DNA byproducts of example 5 and the electrophoretic characterization thereof. Individual bands in fig. 13A: 1-5: 913N agarose gel before recovery by electrophoresis. Individual bands in fig. 13B: 1-2: 913N sepharose recovered self-sheared DNA by-product; 3: 913D control; 4: marker. D represents that the suicide sgRNA and dCas9 expression plasmid co-transform Escherichia coli; n represents suicide sgRNA co-transformed with Cas9N expression plasmid into e.

FIG. 14 is a schematic diagram of the generation of specific linear single strands by self-cleavage of DNA and sequencing primers in example 5.

FIG. 15 is a graphical representation of the sequencing results after the recovery of 913N self-sheared DNA byproduct in example 5.

Fig. 16 is the culture results of the Ori20, 21, 23, 25, 28 target sgRNA expression plasmid and Cas expression plasmid co-transformed escherichia coli in example 6, showing the effect of different targets of the plasmid replicator region on CRISPR/Cas nickase suicide system bacterial culture.

fig. 17 is an agarose gel electrophoresis of products of the mutual transformation of escherichia coli by the Ori20, 21, 23, 25 and 28 target sgRNA expression plasmids and the Cas expression plasmid in example 6, and shows the effect of different targets of the plasmid replicator region on the generation of lsssdna by the CRISPR/Cas nickase suicide system. In the figure, "-" indicates that the plasmid-extracted E.coli was not induced by tetracycline; "+" indicates that the plasmid-extracted E.coli was tetracycline-induced. The PC lane is a plasmid extracted from bacteria that alone transformed the sgRNA expression plasmid; the other lanes are all plasmids extracted from the culture after the suicide sgRNA and the Cas9n expression plasmid are co-transformed into escherichia coli; m is marker.

Fig. 18 is an agarose gel electrophoresis of products of the mutual transformation of escherichia coli with the csi 9n expression plasmid and the Ori2, Ori4, Ori19, Ori22, Ori24, Ori25, Ori27, Ori28 target sgRNA expression plasmids in example 7, showing the effect of different operons on the CRISPR/Cas nickase suicide system for preparing lsssdna. In the figure, "-" indicates that the plasmid-extracted Escherichia coli was not temperature-controlled induced; "+" indicates that the plasmid-extracted E.coli was induced at 42 ℃ with temperature control.

Detailed Description

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

In the present invention, sgRNA is sometimes used in combination with the name of its encoding gene (DNA) for the sake of convenience of description, and those skilled in the art will understand that they represent different substances in different description occasions. For example, the sgRNA gene refers to a gene DNA that can be transcribed into sgRNA.

in the present invention, the terms "linear single-stranded DNA", "single-stranded linear DNA", and "lsssdna" mean the same. Similarly, the terms "linear long single-stranded DNA", "long single-stranded linear DNA", and "long linear single-stranded DNA" mean the same meaning and refer to linear single-stranded DNA.

In the present invention, the terms "Cas nickase transcription expression plasmid", "Cas nickase expression plasmid", "Cas transcription expression plasmid", and "Cas expression plasmid" mean the same meaning. Similarly, the terms "sgRNA gene transcription expression plasmid", "sgRNA gene expression plasmid", "sgRNA transcription expression plasmid", and "sgRNA expression plasmid" represent the same meaning.

In the present invention, "suicide" or "suicide" means that a complex of the sgRNA and the Cas nickase cleaves a single strand or double strand of DNA of a plasmid in which the sgRNA gene itself is present.

The CRISPR/Cas nickase suicide system in the methods of the invention comprises two gene transcription expression elements that are compatible in e.coli cells, wherein one transcription expression element can controllably express a Cas nickase protein and the other expression element is a suicide sgRNA that can be transcribed to a target plasmid controllably or non-controllably.

CRISPR/Cas systems described in the present invention include, but are not limited to, type I, type II and type III CRISPR-Cas systems, and Cas nickase proteins include, but are not limited to, Cas3, Cas9 and Cas 10. In a specific embodiment of the invention, a type II Cas9nickase is preferred, more preferably a streptococcus pyogenes Cas9nickase (spCas9 n). It is well known to those skilled in the art that other nickase forms of Cas proteins can also have similar endonuclease activity and characteristics of Cas9nickase, such as staphylococcus aureus saCas 9nickase, and the like.

The gene transcription expression element in the invention refers to a gene transcription expression element integrated on the genome of Escherichia coli or a gene transcription expression element on a plasmid sequence. The gene transcription expression element comprises gene functional elements such as an operator, a promoter, a target gene sequence, a termination sequence, a replicon and a screening marker gene. Operon (operon) refers to the collective term for a promoter, an operator and a series of closely linked structural genes; in an operon, many functionally related genes are concatenated into a tandem, with a common control region for transcriptional control, including the structural genes and the entire DNA sequence of the regulatory genes. In a specific embodiment of the invention, the operon is capable of regulating the transcriptional expression of the Cas nickase and/or the suicide sgRNA. Transcription regulating expression elements commonly found in prokaryotes include, but are not limited to, the tetracycline operon, the lactose operon, the arabinose operon, the rhamnose regulator, the galactose operon, the pR/pL operon, and the like. In a preferred embodiment of the invention, the lactose operon regulates sgRNA transcription or the J23100 constitutive promoter drives sgRNA transcription. It is well known to those skilled in the art that when the sgRNA gene transcription expression element is non-regulated, its promoter includes, but is not limited to, lacZ, lacI, and J23100 promoters.

The basis of the LssDNA preparation method provided by the invention is as follows: the escherichia coli containing the CRISPR/Cas nickase suicide system is cultured under a non-induction condition, the expression level of the Cas nickase in the suicide system is very low under the regulation of an operator, and the Cas nickase cannot effectively cut DNA under the synergistic effect with sgRNA; when the bacterial culture reaches the logarithmic growth phase, Cas nickase is induced to express, the Cas nickase starts to over-express and assists suicide sgRNA to cut double-stranded DNA of a plasmid where the sgRNA gene is located, and a large amount of plasmid double-stranded DNA forms single-stranded break, namely a nick state; the bacteria are continuously cultured to ensure that the nick plasmid can be replicated and amplified in cells along with the growth of the bacteria, the plasmid needs double-stranded DNA as a template in the theta-type replication process, and LssDNA is formed because one template strand of the plasmid is broken and can not be completely replicated and dissociated, so that the LssDNA can be rapidly prepared in prokaryotes such as escherichia coli.

The target sequence of the suicide sgRNA described in the present invention contains a PAM sequence (protospacer) necessary for recognition of the Cas nickase/sgRNA complex. In a specific embodiment of the invention, the PAM sequence of the spCas 9nickase is the NGG sequence and the target sequence of the suicide sgRNA is the 5 '-Nx-NGG-3' sequence, where N represents any one of bases A, T, C, G and x represents a natural number between 10-30. The suicide sgRNA of the plasmid can target any of the plasmids themselves containing a PAM sequence position, and the cleavage efficiency of Cas9n/sgRNA can be adjusted by increasing or decreasing the target sequence. The suicide sgRNA sequence most suitable for preparing LssDNA can be screened out by using the CRISPR/Cas nickase suicide system. Preferably, the sgRNA obtained by screening has moderate shearing efficiency on the plasmid target sequence, wherein the moderate shearing efficiency means that the Cas nickase protein can be maintained to continuously shear the plasmid, and the plasmid can be maintained at a certain copy number in a cell, so that the plasmid carrying the target gene can be continuously amplified in the cell, and the LssDNA can be generated in a large amount.

In a specific embodiment of the invention, the selection marker genes for the Cas nickase gene transcription expression element and the sgRNA gene transcription expression element are resistance genes, including but not limited to chloramphenicol, ampicillin, kanamycin, and the like.

The Cas nickase transcription expression element and/or the suicide sgRNA transcription expression element described in the present invention are preferably located on a plasmid sequence. In a specific embodiment of the invention, the Cas nickase gene expression element preferably comprises the low copy replicon p15 or psc101, most preferably the replicon is p 15; the sgRNA gene transcription expression element preferably comprises the high copy replicon pMB1 or ColE1, most preferably the replicon is pMB 1. One skilled in the art will appreciate that integration of the Cas nickase or sgRNA expression elements into the e.coli genome or on the same plasmid can also achieve similar transcription or expression functions.

One of the innovation points of the invention is that the DNA endonuclease in the CRISPR/Cas nickase suicide system is a Cas nickase, and the shearing efficiency of the DNA endonuclease depends on the sgRNA target sequence, which is different from the classical restriction endonuclease nickase. The target sequence of sgRNA can be artificially designed and adjusted, and the recognition sequence of the suicide sgRNA targeting plasmid sequence in the CRISPR/Cas nickase suicide system of the invention is about 20 bases generally, so that the Cas nickase only can generate single-stranded damage (nick) at the specific sequence on the plasmid, hardly has influence on the DNA sequence of a bacterial genome, and minimally reduces the influence on the survival of bacteria; classical DNA restriction endonuclease nickases recognize only specific short sequences, typically 4-8 bases, and cannot design or screen plasmid-specific short sequences because of the large size of genomic sequence data. The different target sequences and lengths of the sgrnas result in different cleavage efficiencies of the Cas nickase, and based on this principle, the cleavage efficiency of the sgrnas/Cas nickase can be adjusted, whereas the cleavage efficiency of a classical DNA restriction endonuclease nickase in a cell cannot be adjusted generally.

According to classical bacterial DNA damage repair theory, the CRISPR/Cas nickase suicide system of the present invention may affect bacterial growth, but we find that since the suicide sgRNA of the present invention targets plasmid rather than genomic DNA, neither bacterial growth nor plasmid replication is affected under non-induced conditions. More surprisingly, when partial sgRNA cleavage is "moderately efficient" after induction of Cas nickase expression, double stranded plasmid DNA does not follow classical DNA damage repair mechanisms but produces single stranded DNA by-products that are stable within bacterial cells, similar to phage circular ssDNA, but which are not circular ssDNA but exist in a linear form. The phage ssDNA genome is stable because it can express a protective protein that binds to single-stranded DNA, while plasmid ssDNA is stable in E.coli cells, and has a unique and novel mechanism of formation. By optimizing the culture and induction conditions, the ssDNA byproduct yield is even higher than that of the original plasmid double-stranded DNA. We also found that the efficiency of such ssDNA production depends only on the efficiency of sgRNA cleavage of the target DNA, and not on the position of the target DNA sequence on the plasmid. Experiments have shown that the formation of ssDNA is closely related to the replication process of the plasmid, and the yield of ssDNA is time-dependent. The ssDNA generation mechanism in the invention is completely different from the reported theory, and we believe that in the process of nicked plasmid replication, because Cas nicking enzyme cuts one strand of dsDNA at a fixed point continuously, the replication process taking the broken strand as a template can not be carried out, while the replication of the other complete strand can be completed under the assistance of melting enzyme and replication-related proteins such as SSB and the like, and because ColE1 plasmid replicates in the same direction theta, the broken strand can be dissociated from the double-strand plasmid and forms ssDNA in the incomplete replication process, and finally the production of LssDNA is realized.

Examples

Materials and methods

The whole gene synthesis, primer synthesis and sequencing herein were all accomplished by baio maike biotechnology limited.

Example 1 CRISPR/Cas suicide system Gene transcription expression element and sgRNA target sequence design and construction

In this example, the Cas nickase expression element and/or the suicide sgRNA transcription expression element in the CRISPR/Cas nickase suicide line are located on different plasmid vector sequences, respectively, as a dual plasmid system. The sgRNA expression plasmid in the double-plasmid CRISPR/Cas nickase suicide system is pJ23sgRNA-EGFP, pJ23sgRNA-GFPuv (TacI) or pJ23 TacsgRNA-LacI-GFPuv; cas9n expression plasmids were pETetCas9-Nickase, pPrClCas 9-Nickase or pETetCas9-Nickase (pSC 101). The wtCas9 expression plasmid was pETet-wtCas 9; the dCas9 expression plasmid was pETet-dCas9, and these two plasmids served as control plasmids in this example. All the above-mentioned carrier plasmids are constructed by Baiaomaike biotechnology, Inc., and the nucleotide complete sequences and functional element sequences of all the plasmids are shown in the sequence table. As shown in fig. 2, in the gene transcription expression element, the Cas9n gene and/or sgRNA gene may be transcriptionally expressed by different operons and may comprise different replicons.

1. design of suicide sgRNA target sequence: any sequence containing 5 '-Nx-NGG-3' is selected as a suicide sgRNA target sequence on a suicide target plasmid, and a corresponding DNA primer sequence is synthesized for subcloning test according to the designed suicide sgRNA target sequence as shown in Table 1.

Table 1 different suicide sgRNA target sequences

2. Primers were synthesized from the target sequence as follows:

See table 2 for specific primer sequences.

TABLE 2 primer sequences for each plasmid

Note: f or F in the primer name represents a forward primer, and R or R represents a reverse primer.

3. The plasmid pJ23sgRNA-EGFP was digested with restriction enzymes BamHI and XbaI to recover a linear plasmid DNA, the plasmid pJ23sgRNA-GFPuv (TacI) was digested with restriction enzymes BbaI to recover a linear plasmid DNA, and the plasmid pJ23TacsgRNA-LacI-GFPuv was digested with restriction enzymes BglII and NotI to recover a linear plasmid.

4. When constructing a sgRNA expression plasmid by using pJ23sgRNA-EGFP and pJ23sgRNA-GFPuv (TacI), annealing a synthesized forward and reverse DNA primer to form a sticky end double-stranded DNA, connecting the sticky end double-stranded DNA with the linear plasmid DNA, transforming, and then carrying out PCR identification and screening to obtain a positive clone; when pJ23TacsgRNA-LacI-GFPuv plasmid is used for constructing sgRNA expression plasmid, the sgRNA full sequence is directly synthesized, then the sgRNA full sequence is subjected to double enzyme digestion by restriction enzymes BglII and NotI, and then the sgRNA expression plasmid is connected with linear plasmid DNA for transformation, and positive clone is obtained through PCR identification and screening.

Example 2 uniqueness of LssDNA preparation Using CRISPR/Cas nickase suicide System

1. Test method

The 911F target sgRNA expression plasmid obtained in example 1 and different types of Cas9nickase expression plasmids (Cas9n, wtCas9, dCas9) were co-transformed into Escherichia coli chemical competence, and all transformed bacterial liquid was spread on ampicillin/chloramphenicol double-resistant LB plates and cultured overnight at 37 ℃. The expression plasmids of different Cas9 nickases are pETetCas9-Nickase (p15), pETet-wtCas9 and pETet-dCas9, and the groups of the transforming bacteria are respectively and correspondingly named as 91N, 91WT and 91D.

Selecting positive clones on a resistance plate, respectively culturing in 4mL LB double-resistant culture solution at 37 ℃ for 12 hours, adding a tetracycline inducer before harvesting bacteria so as to induce Cas9 protein to express for 2-4 hours, wherein the induction dose is that 1 mu l of tetracycline hydrochloride solution with the concentration of 10mg/mL is added into each 1mL of LB culture medium, then harvesting all bacteria, extracting plasmids, dissolving in 60 mu l of TE solution, performing 1% agarose gel electrophoresis after extracting the plasmids, and the sampling amount is 2 mu l.

2. Test results

Results as shown in fig. 3, wild-type Cas9+ suicide sgRNA was most efficient at plasmid cleavage, resulting in bacteria that were more sensitive to the corresponding antibiotic screen, and therefore positive clones on the resistance plates were very rare; dCas9 and Cas9Nickase can not cut or can not completely cut the suicide targeting plasmid, so that most plasmids can still replicate and maintain the expression resistance gene, and the number of positive clones is hardly influenced.

In terms of plasmid yield, 91d is greater than 91N, 91N is greater than 91wt, even though the wtCas9 plasmid can exist in a small amount in the cell, the final yield of the plasmid is very low, the bacterial growth speed is lower than that of dCas9 and Cas9Nickase, and particularly after induction, the harvested bacterial quantity is small, and the suicide plasmid almost disappears. In addition, under the induction condition, the yield of the plasmids of the 91d group is increased along with the increase of the culture time; however, since Cas9Nickase still has a partial cleavage effect, the plasmid yield increases slowly.

In agarose electrophoresis FIG. 4, it can be seen that the nicking enzyme group has self-cutting DNA by-product under the plasmid supercoiled DNA, which is linear single-stranded DNA. The CRISPR/Cas nickase suicide system is superior to the CRISPR/wtCas9 suicide system because the CRISPR/wtCas9 suicide system cannot maintain normal replication amplification of the plasmid; the CRISPR/Cas nickase suicide system is superior to the CRISPR/dCas system, and the CRISPR/dCas system cannot realize self-shearing of plasmid DNA, so that the 91D group also generates a linear single-stranded DNA byproduct.

3. Conclusion of the experiment

The CRISPR/Cas nickase suicide system is different from the CRISPR/wtCas9 or CRISPR/dCas suicide system, not only maintains the viability of bacteria with the suicide sgRNA plasmid, but also can keep the replication capacity of the plasmid to a considerable extent. Test results show that the CRISPR/Cas nickase suicide system is a unique novel suicide system and can be used for preparing a DNA self-shearing byproduct, namely linear single-stranded DNA.

Example 3 screening of "moderately efficient" sgRNA targets with CRISPR/Cas9n suicide System

1. Test method

Constructing 491, 911, 107, 140, 385 five target sgRNA expression plasmids according to the method described in the example 1, respectively co-transforming the plasmids and different types of Cas9 expression plasmids (Cas9n, dCas9) into Escherichia coli to be chemically competent, coating ampicillin/chloramphenicol double-resistance LB plates after transformation for overnight culture at 37 ℃, respectively picking a plurality of monoclonal bacteria to be cultured in 4ml double-resistance LB liquid culture medium after the single clone growth is finished, and overnight culture at 37 ℃. Inoculating 200 mu l of the monoclonal strain liquid culture into a new 4mL double-resistance LB liquid culture medium, culturing at 37 ℃, adding 10mg/mL tetracycline hydrochloride liquid inducer according to a proportion of one in a thousand after the monoclonal strain liquid culture medium grows to a logarithmic growth phase to induce the expression of Cas9n, continuously culturing for 6 hours, centrifuging to obtain all bacteria, extracting plasmids, dissolving in 60 mu l of TE solution, extracting the plasmids, and then carrying out 1% agarose gel electrophoresis, wherein the sample dropping amount is 2 mu l. The groups of the transformed bacteria are respectively and correspondingly named as 49N, 49D, 91N, 91D, 107N, 140N and 385N; different clones in the same group are directly renumbered on their group names, e.g., 49N1, 49N2, 49N3, and so on.

2. Test results

The results are as follows: as shown in fig. 5, due to different cleavage efficiencies of sgrnas of different target points, the ratio of supercoiled plasmid/self-cleaved DNA by-product is different, wherein due to higher self-cleavage efficiency of 107, 140 and 385 target points, the number of complete double-stranded DNAs of sgRNA expression plasmid after Cas9n is induced is very small, and a certain amount of resistance genes cannot be expressed, so that the sgRNA expression plasmid cannot grow in double-resistant medium, i.e. cannot extract plasmid. While the target spots 49 and 91 still maintain a certain amount of complete double-stranded DNA due to the weak self-shearing efficiency, so that enough resistance genes can be expressed and can normally grow in a double-resistant LB liquid culture medium, and simultaneously, a large amount of small-molecular-weight self-shearing DNA byproducts are generated under the synergistic action of the shearing of Cas9n and the self-replication of plasmids. Preferably, 491 and 911 target points are used as sgRNA targets for further optimization. Wherein, the 49 # target point can generate a certain amount of self-shearing DNA by-products at the Cas nickase leakage expression level, and the plasmid replication is influenced after the Cas nickase is induced to express; whereas target point 91 produces more self-sheared DNA by-products after Cas nickase-induced expression.

3. Conclusion of the experiment

The result shows that the sgRNA target with moderate shearing efficiency can be stably and quickly screened out by the CRISPR/Cas nickase suicide system.

Example 4 adjusting sgRNA target length to screen optimal "moderate efficiency" sgRNA

1. Test method

According to the test result in the above example 3, the 911 and 491 target points are respectively lengthened or shortened by several bases, the corresponding suicide sgRNA expression vectors are reconstructed and named as 912, 913, 914, 492, 493 and 494 target points, and then the suicide sgRNA expression vectors and the Cas nickase expression plasmids are co-transformed into escherichia coli. The induction test method, the detection method and the grouping naming rule are the same as those in example 3. Wherein the lengths of the 491-; the lengths of the sgRNA targets of 911-one 914 are 20, 17, 24 and 27nt respectively.

2. Test results

As shown in FIG. 6, the cleavage efficiencies of the 491-494 series target and the 911-914 series target both showed strong target length dependence. Compared with 491 series targets with stronger shearing efficiency, the plasmid replication capacity of 492 and 493 targets with shortened target length is better, and the yield of DNA self-shearing byproducts is higher; when the length of the No. 494 target point is extended to 25nt, the shearing efficiency is strongest and bacteria can not grow normally in the double-resistant culture medium, so that the corresponding plasmid can not be extracted. The 911-914 series of targets also exhibited enhanced shear efficiency with increasing target length. Because the cleavage efficiency depends not only on the sgRNA length but also on the base composition in the sequence, the target showed moderate cleavage efficiency even when the 914 target was elongated to 27 nt. The target points 913, 914, 492 and 493 are preferably the optimal sgRNA target points.

3. Conclusion of the experiment

The self-shearing capacity of the sgRNA gene expression plasmid in a CRISPR/Cas nickase suicide system can be adjusted by lengthening or shortening the length of the sgRNA target sequence, and the target sequence which is most suitable for producing linear single-stranded DNA and has moderate shearing efficiency is screened out, so that the yield of self-sheared DNA byproducts is increased.

Example 5 identification of self-sheared DNA product of CRISPR/Cas nickase suicide System as Linear Single-stranded DNA

1. Test method

a) and (3) molecular weight size determination: constructing a sgRNA expression vector pJ23TacsgRNAOri1R-LacI-GFPuv targeting Ori1R target in addition to the sgRNA expression vectors of 491-; then, self-sheared DNA byproducts of different targets were produced and purified as described in example 3; the molecular weight of DNA self-sheared DNA byproducts of different samples was analyzed by 1% agarose gel electrophoresis, the sample size was 3. mu.l, and the agarose gel dye was Ethidium Bromide (EB).

b) SYBR Green I DNA staining and acridine orange DNA staining: 1% agarose of SYBR Green I (Bai' ao Mei Ke) and Gold view (assist holy Boss, acridine orange dye) was prepared according to the product specification for gel electrophoresis, the samples were the self-sheared DNA products of different targets in step a), the amount of the spots was 3. mu.l, and the images were developed in an ultraviolet gel imager.

c) And (3) restriction enzyme digestion detection: respectively shearing self-sheared DNA products of different suicide sgRNA targets according to type II DNA restriction endonucleases EcoRI, PstI and HpaII product specifications (NEB), and then carrying out agarose gel electrophoresis analysis, wherein all samples are the self-sheared DNA products of different targets in the step a).

d) Degradation detection of the single-strand specific nuclease S1: the self-sheared DNA products of the different suicide sgRNA targets were digested according to nuclease S1(Invitrogen) instructions and analyzed by agarose gel electrophoresis, the samples being the self-sheared DNA products of the different targets in step a). The agarose gel electrophoresis dye is acridine orange.

e) sequencing and verifying: and (3) recovering the self-shearing DNA by-product of the suicide sgRNA target according to a DNA gel recovery kit (Biomics), detecting through agarose gel electrophoresis, sequencing and analyzing a sequencing result.

2. Test results

a) As shown in fig. 7, the CRISPR/Cas nickase suicide system can allow sgRNA expression plasmids with completely different sequences to all be generated from the sheared DNA byproduct. Co-transformation of E.coli with the dCas9 expression plasmid does not result from shearing of the DNA by-product.

In the test, sgRNA expression plasmids of No. 492 and No. 913 targets are both pJ23sgRNA-EGFP, the total length of the sequences is 3.7kb, and the plasmids are ampicillin resistance; the sgRNA expression plasmid of the OriR1 target spot is pJ23TacsgRNA-LacI-GFPuv, and the total length of the sequence is about 5.2 kb; the plasmid pETetCas9-Nickase (p15) sequence is about 6.7kb in total length. As shown in FIG. 8, the self-sheared DNA products were re-electrophoresed and compared with the 1kb DNA marker of Tiangen Biochemical technology Ltd to calculate the sizes of the self-sheared DNA by-products of different plasmids. The results show that the molecular weights of the self-sheared DNA byproducts of different plasmids are equal to 1/2, namely 1.9kb, 1.9kb and 2.6kb, of the molecular weight of the original suicide sgRNA expression plasmid, so that the self-sheared DNA byproducts are identical to the above experimental results when the self-sheared DNA byproducts are complete plasmid single-stranded DNA.

b) SYBR Green I is a dye with a Green excitation wavelength that binds to all dsDNA duplex minor groove regions. In the free state, SYBRGreen I emits weak fluorescence, but once bound to double-stranded DNA, fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of SYBR Green I is related to the quantity of double-stranded DNA, and the quantity of the double-stranded DNA existing in the PCR system can be detected according to the fluorescence signal. SYBRGreen I has a maximum absorption wavelength of about 497nm and an emission wavelength of about 520 nm. The maximum excitation wavelength is 454 nm. Under ultraviolet radiation perspective, SYBR Green I bound to double stranded DNA appears Green fluorescent. If the agarose gel contains single-stranded DNA, the color is orange rather than green.

SYBR Green I agarose gel electrophoresis is carried out on self-shearing DNA byproduct samples of different suicide sgRNA expression plasmids, and the detection is observed under an ultraviolet lamp. As shown in fig. 9: cas nickase and sgRNA expression plasmid are double-stranded closed-loop DNA, both supercoiled and open-loop plasmids are green, dCas9 cannot cut the sgRNA plasmid, and a corresponding lane has no orange ssDNA band; cas9n cleaves sgRNA expression plasmid and results in a decrease in dsDNA morphology, weakening of green supercoiled and open circular plasmid bands, and the lowest more self-cleaving DNA byproduct band as orange ssDNA.

Acridine Orange (AO) belongs to a tricyclic heteroaromatic dye, and can stain dsDNA, ssDNA, or RNA. There are two main ways of binding nucleic acids: one is insertion combination, AO is inserted between base pairs of nucleic acid double-chain, the mode is that AO is combined with dsDNA, its fluorescence emission peak is 530nm, it is green fluorescence after excitation; the other is electrostatic attraction, the attraction and combination between the AO with positive charge and the phosphate radical (with negative charge) of the single-stranded nucleic acid are generated, the combination mode is mainly the combination of the AO, ssDNA and RNA, the fluorescence emission peak is 640nm, the fluorescence is red after excitation, and a small amount of combination can turn orange yellow or orange red. Therefore, acridine orange is embedded into a double-stranded DNA molecule to show green color, and emits orange or orange red fluorescence when being combined with a single-stranded DNA or RNA.

As shown in fig. 10: co-transformation of dCas9 and suicide sgRNA failed to produce ssDNA that developed red in color; whereas co-transformation of Cas9n and the suicide sgRNA can produce a large amount of self-sheared ssDNA by-product that develops red in color; other morphologies of both sgRNA and Cas9 expression plasmids appear green.

c) As shown in fig. 11A: EcoRI and PstI are used for single enzyme digestion to identify self-cutting DNA products of different groups, 491D and 913D are completely consistent with 491N and 913N theoretical digestion products, but the self-cutting DNA by-products cannot be digested by restriction endonucleases capable of recognizing double-stranded DNA. As shown in FIG. 11B, HpaII restriction enzyme was selected and tested, and the self-cleavage by-product was still not digested for a certain period of time, while the plasmid in other forms was completely digested. Unlike EcoRI and PstI, the HpaII enzyme recognizes only four bases "CCGG" of double-stranded DNA, and thus up to 18 digestion sites are uniformly distributed on the suicide sgRNA expression plasmid. In the presence of so many recognition sites, the self-cutting DNA by-product remains stable, indicating that the sequence in these recognition site regions is in single-stranded DNA form.

d) Nuclease S1 is derived from Aspergillus oryzae (Aspergillus u.soryzae), and is a highly single-stranded specific endonuclease that specifically degrades single-stranded DNA or RNA under optimal enzymatic reaction conditions to produce single nucleotides or oligonucleotides with 5' -phosphate. The enzyme is relatively insensitive to double-stranded DNA, double-stranded RNA and DNA-RNA hybrids. Nuclease S1 hydrolyzes single-stranded DNA 75000 times faster than double-stranded DNA. The single-strand hydrolysis function of S1 nuclease can act on the single-stranded region of a double-stranded nucleic acid molecule and cleave the nucleic acid molecule from the single-stranded site, and this single-stranded region can be as small as only one base pair, so that the nuclease S1 can be used to discriminate dsDNA from ssDNA.

As shown in fig. 12: the self-sheared DNA byproduct can be completely degraded by nuclease S1, no red ssDNA band exists in the corresponding position of 491N and 913N enzyme digestion group lanes, and ssDNA red bands exist in the non-enzyme digestion group; the 491D control group had no red ssDNA band regardless of digestion, since nuclease S1 did not degrade double-stranded DNA, but could still form DNP (DNA protein complex) causing the position of dsDNA with different morphology to move up. The above results indicate that the self-sheared DNA byproduct is completely single stranded.

e) As shown in FIG. 13, the 913N self-sheared DNA byproduct can be successfully recovered using an agarose gel electrophoresis recovery kit. Sequencing the ssDNA recovery product, and respectively setting forward primers as M13F: 5'-TGTAAAACGACGGCCAGT-3'), eGFP-F: 5'-CATGGTCCTGCTGGAGTTCGTG-3'), the reverse primer is M13R: 5'-CAGGAAACAGCTATGACC-3').

As shown in fig. 14, to ensure that sequencing can verify whether the self-sheared DNA byproduct is linear ssDNA, the sequence of sequencing primers M13F and M13R is less than 800bp from the Cas nickase cleavage site to ensure that the sequencer can detect the site signal; primer eGFP-F is used to check the integrity of the linear single strand, and its sequence is located at the 3' end of the predicted linear ssDNA.

The results are shown in FIG. 14, where both forward primers were able to sequence normally, while the reverse primer was unable to sequence successfully. It was demonstrated that Cas nickase Cas9(D10A) used in this experiment specifically cleaves a single DNA strand complementary to the sgRNA, which single strand nicks on the antisense strand, resulting in the generation of self-sheared ssDNA product as antisense strand, and thus the reverse primer was not sequenced successfully.

As shown in fig. 15, sequencing with the M13F primer ended with the Cannes at the theoretical cleavage site of Cas9n, i.e., 4 bases upstream of the PAM site. The above results indicate that Cas9n (D10A) cleaves the sgRNA complementary strand and causes the single strand to separate from the plasmid, forming a self-cleaving DNA byproduct that is linear ssdna (lsssdna); the LssDNA 5' end sequence is complete and has no base degradation in cells, which is probably related to the binding protection of replication-related proteins; successful sequencing of the eGFP-F primer near the 3 'end indicated that the ssDNA sequence was also intact at the 3' end.

3. Conclusion of the experiment

The self-cutting DNA byproduct generated by the CRISPR/Cas nickase suicide system is linear single-stranded DNA (LssDNA), the single strand is a Cas nickase cutting strand separated from plasmid dsDNA, and the sequences at both ends of the linear single-stranded DNA are not degraded completely.

example 6 Generation of LssDNA by CRISPR/Cas nickase suicide System as non-sequence-specific

1. test method

Different sgRNA expression plasmids targeting the oriV replication sequence of the plasmid, target sequences and primers were constructed as described in example 1, see example 1. The constructed suicide sgRNA expression plasmid and the Cas nickase plasmid are co-transformed into escherichia coli competence. The induction test method, the detection method and the grouping naming rule are the same as those in example 3. The sgRNA expression plasmid is pJ23sgRNA-GFPuv (TacI), the plasmid carries a GFPuv gene expression frame, and the quantity of GFPuv expression can be evaluated by observing the quantity of green fluorescent protein in an escherichia coli culture solution under UV rays. Detecting the quantity of LssDNA generated by different sgRNA targets in a CRISPR/Cas nickase suicide system; it was evaluated whether or not LssDNA production correlated with the expression level of GFPuv. PC is plasmid expression bacteria only transforming pJ23sgRNA-GFPuv (TacI), NC is plasmid-free Escherichia coli.

2. Test results

As shown in fig. 16, after Cas nickase induced expression, strains transformed with Ori20, 21, 23, 25, 28 target plasmids were much lower than the PC group in both GFPuv expression level and bacterial culture concentration. The expression amount of the target GFPuv of Ori20 and Ori28 and the concentration of the bacterial culture are the lowest, the target of Ori21 and Ori23 is better than the target of Ori20 and Ori28, and the target of Ori25 is the best. The expression level of GPFuv is in positive correlation with the concentration of the bacterial culture.

Plasmids were extracted after each induction and the results are shown in FIG. 17: 21. the 23 and 25 target plasmids can induce the generation of ssDNA products, but the 25 target shearing efficiency is moderate, and the yield is highest; after the No. 20 and 28 target points are induced, the bacteria grow very little and plasmids cannot be extracted, and the sgRNA expression plasmids and the sheared DNA byproducts in the plasmids extracted from the bacterial culture before induction are also very little. The quantity of LssDNA generated by self-shearing is in positive correlation with the expression quantity of GFPuv and the concentration of bacterial culture.

In this example, each target sequence is located in the replicator region (Ori) and does not target the GFPuv expression gene or the ampicillin resistance gene, and the CRISPR/Cas nickase suicide system does not act directly on the expressed gene elements, and both the expression level of GFPuv and the number of bacterial cultures depend on the copy number of the sgRNA expression plasmid in bacteria. Test results show that the generation amount of LssDNA by-products is only related to the shearing efficiency of the used sgRNA target, and when the shearing efficiency is high, the copy number of the corresponding suicide plasmid is reduced, so that the expression amount of GFPuv and ampicillin resistance genes is directly reduced, and the growth of bacteria is influenced. The generation of LssDNA is favored when the shearing efficiency is moderate.

3. Conclusion of the experiment

The generation mechanism of LssDNA in the CRISPR/Cas nickase suicide system is non-sequence specificity, is only related to the shearing efficiency, is not related to the position of a target sequence in a plasmid, and the shearing efficiency is closely related to the base composition and the sequence length of a target spot.

example 7 Effect of different operons on the preparation of LssDNA by CRISPR/Cas nickase suicide System

1. Test method

Constructing sgRNA expression plasmids of Ori2, Ori4, Ori19, Ori22, Ori24, Ori25, Ori27 and Ori28 according to the method described in example 1, co-transforming the plasmids with Cas9n expression plasmid pPrPl Cas9Nickase to be chemically competent, coating ampicillin/chloramphenicol double-resistant LB plates after transformation, culturing overnight at 37 ℃, and selecting several monoclonals to culture in 4ml double-resistant LB liquid culture medium overnight at 37 ℃ after the monoclonals grow. Inoculating 200 mu l of the monoclonal strain liquid culture into a new 4ml double-resistance LB liquid culture medium for culture at 37 ℃, adjusting the culture temperature to 42 ℃ after the culture reaches the logarithmic growth phase to induce the expression of Cas9n, continuously culturing for 4 hours, centrifuging to obtain all bacteria, extracting plasmids, dissolving the plasmids in 60 mu l of TE solution, extracting the plasmids, and then carrying out 1% agarose gel electrophoresis, wherein the sample amount is 2 mu l.

2. Test results

The operon in a transcription expression element of the Cas nickase gene in the CRISPR/Cas nickase suicide system is replaced by a PrPl temperature control operon, and whether the preparation of linear single-stranded DNA is influenced by different operons is verified. The plasmid pETetCas9-Nickase is replaced by plasmid pPrpl Cas9Nickase, and the induction condition is changed into 42 ℃ for culture. As shown in fig. 18, in the new CRISPR/Cas nickase suicide system, suicide sgrnas of different targets still readily form self-cleavage by-product lsssdna. Similar to the tetracycline operator CRISPR/Cas nickase suicide system, different suicide sgrnas have different cleavage efficiencies, and the yields and the proportion of lsssdna in the total DNA are different.

3. Conclusion of the experiment

In a CRISPR/Cas nickase suicide system, the gene transcription expression element can use different types of operons to regulate and control the transcription expression of Cas nickase and/or sgRNA, and the purpose of preparing LssDNA can be achieved.

the above examples demonstrate the technical scheme of preparing linear single-stranded DNA (LssDNA) according to the present invention, and it will be understood by those skilled in the art that various changes or modifications may be made on the basis of the above examples without departing from the spirit of the present invention, and the equivalents of the various changes or modifications should also fall within the scope of the present invention.

Claims

1. A method for preparing linear single-stranded DNA in Escherichia coli, comprising the steps of:

A) Constructing a Cas expression element capable of expressing Cas nickase, wherein the Cas expression element comprises an operator, a promoter, a Cas nickase gene sequence, a terminator, a replicon and a screening marker gene, and the Cas nickase is spCas9 derived from streptococcus pyogenes or sacAS9 derived from staphylococcus aureus;

B) Integrating the Cas expression element obtained in the step A) into a plasmid or a genome to obtain a Cas nickase expression plasmid;

E) Co-transforming the sgRNA expression plasmid obtained in step D) and the Cas nickase expression plasmid obtained in step B) into an Escherichia coli competent cell;

2. The method of claim 1, wherein the Cas nickase is Cas9(D10A) shown as SEQ ID No:1 or Cas9(H840A) shown as SEQ ID No: 2.

3. The method of claim 1, wherein said PAM sequence is a5 '-Nx-NGG-3' sequence, wherein N represents any one of bases A, T, C, G and x represents any natural number between 10 and 30.

4. The method of claim 1, wherein the operons in the Cas expression element and the sgRNA gene transcription expression element are each independently selected from the group consisting of: tetracycline operon, lactose operon, galactose operon, arabinose operon, rhamnose regulator, pR/pL temperature control operon.

5. The method of claim 1, wherein the selectable marker genes in the Cas expression element and the sgRNA gene transcription expression element are each independently selected from the group consisting of: chloramphenicol resistance gene, ampicillin resistance gene, kanamycin resistance gene.

6. The method of claim 1, wherein the promoter in the sgRNA gene transcription expression element is selected from the group consisting of: TacI, LacZ, LacI, J23100.

7. The method of claim 1, wherein the replicon in the sgRNA gene transcription expression element is selected from pMB1 and ColE 1; the replicon in the Cas expression element is selected from p15 and pSC 101.

8. The method of claim 1, wherein the Cas nickase expression plasmid is selected from the group consisting of: pETetCas9-Nickase shown in SEQ ID No. 3, pETetCas9-Nickase (pSC101) shown in SEQ ID No. 4, and pPrpl Cas9Nickase shown in SEQ ID No. 5.

9. The method of claim 1, wherein the sgRNA expression plasmid is selected from the group consisting of: pJ23sgRNA-EGFP shown in SEQ ID No. 6 and pJ23sgRNA-GFPuv (TacI) shown in SEQ ID No. 7.

10. A kit for preparing linear single-stranded DNA, comprising: a Cas nickase expression plasmid comprising a Cas expression element that can express a Cas nickase; a sgRNA expression plasmid comprising a sgRNA gene transcription expression element compatible with the Cas expression element described above in an escherichia coli cell; and host E.coli; the Cas Nickase expression plasmid is selected from pETetCas9-Nickase shown in SEQ ID No. 3, pETetCas9-Nickase (pSC101) shown in SEQ ID No. 4 and pPrPl Cas9Nickase shown in SEQ ID No. 5; the sgRNA expression plasmid is selected from pJ23sgRNA-EGFP shown in SEQ ID No. 6 and pJ23sgRNA-GFPuv (TacI) shown in SEQ ID No. 7.