CN120683099A

CN120683099A - An efficient gene editing system for Streptomyces and its construction method and application

Info

Publication number: CN120683099A
Application number: CN202410603447.1A
Authority: CN
Inventors: 童垚俊; 罗晶; 谢苡恩
Original assignee: Shanghai Jiao Tong University
Current assignee: Shanghai Jiao Tong University
Priority date: 2024-05-15
Filing date: 2024-05-15
Publication date: 2025-09-23

Abstract

The present invention discloses a high-efficiency gene editing system for Streptomyces and its construction method and application; the system includes a TnpB nuclease and a guide RNA that can be expressed in Streptomyces, and the guide RNA includes an RNA backbone, a gene targeting segment, and a gene sequence of a hepatitis delta virus (HDV) ribozyme. Wherein, the gene targeting segment is located at the 3' end of the RNA backbone and is a nucleic acid fragment with a length of 12-40bp after the TAM sequence (5'TTGAT) on the target gene; the hepatitis delta virus (HDV) ribozyme is used to stabilize the RNA backbone-gene targeting segment structure. The Streptomyces mini gene editing tool provided by the present invention can achieve a gene editing efficiency of 70-100% in Streptomyces in the presence of a homologous repair template, wherein the gene editing efficiency under the action of the rationally designed guide RNA can reach 100%.

Description

Efficient gene editing system for streptomycete, and construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and relates to a high-efficiency gene editing system for streptomycete, and a construction method and application thereof. In particular to an optimized guide RNA and TnpB gene editing system, a construction method thereof and application thereof in streptomycete. The invention develops a novel mini gene editing tool applicable to streptomycete by optimizing the guide RNA of TnpB system. The streptomycete minigene editing tool can realize accurate and efficient editing of genome in streptomycete body, and has great popularization and application values.

Background

Streptomyces (Streptomyces) is the largest genus among actinomycetes, and is considered to be a very development-valuable group because it produces a large number of valuable active secondary metabolites and also contains abundant silent biosynthetic gene clusters in its genome. From genome information, the efficient high-throughput development of novel active secondary metabolites based on the concept of synthetic biology from bottom to top is a main stream idea of the current natural product drug development, and the key is to have an efficient, accurate and convenient genetic operation system. However, streptomyces has a large genome (8-10 Mb) and a high GC content (generally, GC content is more than 70%), and compared with other microorganisms, genetic manipulation such as gene editing is difficult, and genetic manipulation means are limited.

With the development of a CRISPR-Cas gene editing system and the application of the CRISPR-Cas gene editing system in streptomyces, the problem of time-consuming and low-efficiency streptomyces gene editing is relieved to a great extent, the bottleneck of lack of efficient genetic operation in the field of streptomyces is effectively broken, the CRISPR-Cas gene editing system becomes a main gene editing tool currently applicable to streptomyces, efficient and accurate gene editing of streptomyces genome is realized, and the CRISPR-Cas gene editing system is well applied in the aspects of exploring new secondary metabolites of streptomyces, improving secondary metabolism yield, improving metabolism pathways and the like. However, there are still many problems to be solved in current CRISPR-Cas system based gene editing, such as Cas9 or Cas12a proteins with more than 1000 amino acids, i.e. with more than 3000 nucleotides (pairs) encoding such effector proteins, and efficient packaging of such many nucleotides into some delivery systems is difficult, thus affecting plasmid construction and transformation efficiency. The bulkiness of Cas9/Cas12a also limits the retrofit space of subsequent editing systems.

In order to break through the application limitation caused by large protein volumes such as Cas9, researchers have studied and explored from different directions to overcome the defects by modifying the CRISPR-Cas system through system optimization, such as modifying Cas9, optimizing guide RNA, using Cas9 orthologous enzyme and a series of measures, exploring and applying novel gene editing technology such as developing PRIME EDITING and the like, developing a novel CRISPR system, excavating and developing compact CRISPR proteins, including CasX (about 980 amino acids), cas12f (400-700 amino acids), cas12I (about 1000 amino acids), cas12 phi (700-800 amino acids), cas12m (604 amino acids), cas12I (about 860 amino acids), caslambda (about 800 amino acids) and the like. The TnpB protein is a programmable nuclease discovered in transposon systems in recent years, is evolutionarily likely to be an ancestor of Cas12, can be guided by long non-coding RNAs of about 150nt to cleave DNA sequences near the 5' end of TTGAT, and has a volume of only about 1/3 (about 400 amino acids) of Cas protein, and is of interest to researchers at home and abroad. To date, tnpB nucleases have been successfully used for endogenous gene editing in human cells, mouse embryos, monocots, dicots, and the like. Researchers have also performed extensive and systematic mining and research of TnpB nucleases widely distributed in organisms to identify a variety of TnpB with targeted editing activity. However, in general, tnpB has low editing efficiency compared to Cas9, and more importantly, the existing small-sized editors have not been reported to perform gene editing in streptomycete.

Disclosure of Invention

The invention aims to provide a high-efficiency gene editing tool for streptomycete, and a construction method and application thereof. Aiming at the current situation that a mini gene editing tool is not available in streptomyces and the problem that the current mini gene editing tool is generally low in editing efficiency, a core element for realizing gene editing by deeply analyzing small-volume programmable TnpB nuclease and an action mechanism thereof are designed, an RNA sequence for guiding TnpB nuclease to realize double-chain cutting is designed, and the system is developed to be suitable for the efficient mini gene editing tool of the streptomyces, so that efficient and accurate editing of the genome of the streptomyces based on double-chain fracture and directional repair is realized.

The invention aims at realizing the following technical scheme:

In a first aspect, the invention provides a guide RNA for directing the movement of TnpB proteins towards a target sequence. The guide RNA (in sequence in the direction of the 5 'to 3' end) comprises an RNA backbone, a gene targeting segment, and a gene sequence of Hepatitis Delta Virus (HDV) ribozyme.

In one embodiment, the nucleotide sequence of the RNA backbone is SEQ ID No.3.

In one embodiment, the nucleotide sequence of the RNA backbone is a segment of the RNA nucleotide sequence rationally designed by the deep research analysis of TnpB guide RNA, and the nucleotide sequence is SEQ ID NO.6.

In the present invention, the gene targeted by the system is the gene located in or near the TAM sequence (5' TTGAT). In one embodiment, the gene targeting segment, located at the 3 'end of the RNA backbone, is a nucleic acid fragment of 12-40bp in length after the TAM sequence (5' ttgat) on the target gene. The Hepatitis Delta Virus (HDV) ribozymes are useful for stabilizing RNA backbone-gene targeting segment structures.

In a second aspect, the invention provides a TnpB-mediated Streptomyces minigene editing system comprising a TnpB nuclease expressible in Streptomyces and the guide RNA.

In one embodiment, the TnpB nuclease is a TnpB nuclease that is codon optimized for expression in streptomyces (DNA similarity to TnpB from which wild-type Deinococcus radiodurans ISDra2 is derived is 79.74%). The amino acid sequence of TnpB nuclease is shown as SEQ ID NO. 1. The gene sequence of the TnpB nuclease is shown as SEQ ID NO. 2.

In a third aspect, the present invention provides a recombinant expression plasmid vector for expressing the Streptomyces minigene editing system.

In a fourth aspect, the present invention provides a method for constructing the Streptomyces (high efficiency) minigene editing system, comprising the steps of:

the construction comprises the step of carrying out gene knockout/knock-in of a screening marker containing apramycin resistance, wherein the gene knockout/knock-in plasmid is formed by respectively inserting TnpB protein subjected to codon optimization and guide RNA and gene knockout/knock-in boxes thereof on the basis of an escherichia coli-streptomycete shuttle plasmid.

In some implementation examples, the coding-optimized TnpB protein and the guide RNA thereof are respectively substituted for Cas9 and sgRNA fragments on a streptomycete-escherichia coli shuttle plasmid to obtain a plasmid A, the plasmid A is digested to obtain a linearized plasmid B, and the gene knockout/knock-in plasmid is obtained by inserting the gene knockout/knock-in box.

In one embodiment, the gene knockout plasmid is based on a shuttle plasmid of escherichia coli-streptomyces, and TnpB gene editing system (TnpB protein and guide RNA thereof) and gene knockout cassette suitable for streptomyces are inserted.

In one embodiment, the gene knockout cassette comprises the core elements of an upstream homology arm of the target gene and a downstream homology arm of the target gene in sequence in the direction of editing the target gene, wherein the upstream and downstream homology sequences of the target gene are used for providing a homologous recombination template when gene editing occurs.

In one embodiment, the gene knock-in plasmid is based on a shuttle plasmid of E.coli-Streptomyces, and a TnpB gene editing system (TnpB protein and guide RNA thereof) and a gene knock-in cassette suitable for Streptomyces are inserted.

In one embodiment, the gene knock-in cassette comprises core elements of a homology arm upstream of the target gene, an insert gene, and a homology arm downstream of the target gene in that order in the direction in which the target gene is edited.

In a fifth aspect, the invention provides an application of the streptomycete minigene editing system in precise editing of a streptomycete genome based on double strand break and directed repair.

In a sixth aspect, the present invention provides a method for gene editing of a target gene in Streptomyces recipient using the above Streptomyces minigene editing system, the method comprising:

① Under the non-induction condition, transforming the gene knockout/entry plasmid constructed by the method into a target host, realizing gene knockout (entry) by homologous exchange of a homologous arm sequence of the gene knockout/entry box and an upstream and downstream homologous sequence of a target gene, and obtaining a transformant carrying the gene knockout/entry plasmid through screening of antibiotics with plasmid related resistance;

② Streaking the transformant in the step ① to a culture medium plate containing plasmid-resistant antibiotics and promoter inducers, and culturing at constant temperature until the monoclonal antibody is visible;

③ Randomly selecting a step ② monoclonal, and performing colony PCR verification to obtain a traceless gene knockout/entering strain.

In some embodiments of the invention, the promoter-inducer is selected from the group consisting of thiostrepton.

Compared with the prior art, the streptomycete mini gene editing tool provided by the invention can achieve 70-100% of gene editing efficiency in streptomycete under the existence of the homologous repair template, wherein the gene editing efficiency under the action of the guide RNA after rational design can reach 100%.

Drawings

Other features, objects, advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:

FIG. 1 is a map of HpTnpB-reRNA-ZH and HpTnpB-reRNA-ZJ plasmids;

FIG. 2 is a schematic diagram of the genome structure of a strain in which a target gene (SCO 5087) was successfully edited theoretically;

FIG. 3 is a diagram showing the result of editing the endogenous gene of Streptomyces (DNA electrophoresis) by the TnpB system (HpTnpB-reRNA-ZJ) of the present invention;

FIG. 4 is a comparison of the efficiency of editing of endogenous genes of Streptomyces by HpTnpB-reRNA-ZH and HpTnpB-reRNA-ZJ;

FIG. 5 is a graph of Sanger sequencing results.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that several modifications and improvements can be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.

The embodiment of the invention takes streptomycete model strain Streptomycin coelicolor A (2) as a target strain, and the genome sequence number of the strain is GeneBank:GCA_008931305.1. According to the embodiment of the invention, a key gene SCO5087 (actI) synthesized by actinorhodin in the strain is taken as an endogenous gene target, gene knockout is taken as a target, and the editing efficiency of the invention is tested.

EXAMPLE 1 construction of Streptomyces efficient Mini gene editing tool

1.1 Plasmid design and construction

The TnpB gene is a TnpB gene with optimized codons, the codon optimization is carried out on the TnpB protein coding gene according to the codon preference of streptomycete, the amino acid sequence is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2. A guide RNA, designated reRNA-ZH, comprises, in order from the 5 'to the 3' end, the core elements of an RNA backbone (SEQ ID NO. 3) which is a sequence directing TnpB cleavage of double-stranded DNA near the target gene (https:// doi. Org/10.1038/s 41586-021-04058-1), a gene targeting segment (sequence as shown in SEQ ID NO.4 when SCO5087 as described above is the target) and the gene sequence of Hepatitis Delta Virus (HDV) ribozyme (SEQ ID NO. 5). Another guide RNA, designated reRNA-J, comprises, in order from the 5 'to the 3' end, the following core elements, an RNA backbone (SEQ ID NO. 6), a gene targeting segment (sequence shown as SEQ ID NO.4 when SCO5087 as described above is the target point), and a gene sequence of Hepatitis Delta Virus (HDV) ribozyme (SEQ ID NO. 5), wherein the RNA backbone (SEQ ID NO. 6) is a segment of the RNA nucleotide sequence rationally designed by the present invention. The above gene fragments were synthesized by Kirschner Biotech Co., ltd.

The TnpB gene after codon optimization and reRNA-ZH replace Cas9 and sgRNA fragments on Streptomyces coli shuttle plasmid vector pCRISPR-Cas9 (https:// doi.org/10.1021/acssynbio.5b00038) respectively to obtain plasmid pTnpB-reRNA-ZH.

The TnpB gene after codon optimization and reRNA-J are respectively substituted for Cas9 and sgRNA fragments on a Streptomyces-E.coli shuttle plasmid vector pCRISPR-Cas9 (https:// doi.org/10.1021/acslynbio.5b00038) to obtain a plasmid pTnpB-reRNA-ZJ.

Then plasmid pTnpB-reRNA-ZH and pTnpB-reRNA-ZJ were digested with SphI endonuclease to obtain linearized pTnpB-reRNA-ZH and linearized pTnpB-reRNA-ZJ. Then, the gene knockout boxes formed by the upper and the lower homologous arms of the target gene are respectively assembled with linearization plasmid vectors pTnpB-reRNA-ZH and pTnpB-reRNA-ZJ in a seamless cloning manner (the kit is purchased from Vazyme and ClonExpress Ultra One Step Cloning Kit), so as to obtain plasmids HpTnpB-reRNA-ZH (the plasmid mass spectrogram is shown in figure 1) and HpTnpB-reRNA-ZJ (the plasmid spectrogram is shown in figure 1). When SCO5087 (actI) is taken as an endogenous gene target point, the upstream homology arm sequence is shown as SEQ ID NO.7, and the downstream homology arm sequence is shown as SEQ ID NO.8. The present invention performed Sanger sequencing on all constructed plasmids to ensure complete correctness.

Example 2 application of Streptomyces high-efficient Mini-editing System

2.1 Conversion

The plasmid of interest was transferred into E.coli ET12567/pUZ8002 (https:// doi.org/10.1016/0378-1119 (92) 90549-5) by adding 200ng of plasmid HpTnpB-reRNA-ZH or HpTnpB-reRNA-ZJ, respectively, to 100. Mu.L of thawed self-made competent cells E.coli ET12567/pUZ8002, mixing the walls of the flick tube, standing on ice for 30 minutes, then standing in a 42℃water bath for 45 seconds, then immediately placing on ice for 2-3 minutes, then adding an antibiotic-free LB liquid medium, 200rpm,37℃for 1 hour, then centrifuging at 5000rpm for 5 minutes, discarding 900. Mu.L of supernatant, adding LB solid plates containing 25. Mu.g/mL, 12.5. Mu.g/mL chloramphenicol and 50. Mu.g/mL of amphotericin, after culturing at 37℃overnight, picking up single clone to 20mL of kanamycin (25. Mu.g/mL), adding antibiotic-free LB liquid medium at 200rpm,37℃for about 4 minutes, centrifuging at 5000. Mu.g/mL, and 5 rpm repeatedly adding the antibiotic-free LB liquid medium at about 4 rpm, and centrifuging at about 4 rpm for 20 minutes.

2.2, Binding transfer and resistance screening

Under non-induction conditions, the plasmids in step 2.1 are transferred into Streptomyces coelicolor A (2) through combination, the specific method comprises the steps of taking streptomycete spores collected in advance, centrifuging at 5000rpm for 5 minutes, discarding the supernatant, resuspending thalli with 2mL of 2 XYT liquid culture medium, placing a centrifuge tube filled with spores in a 50 ℃ water bath kettle for heat shock for 10 minutes, then pre-germinating for 30 minutes in a 30 ℃ shaker at 200rpm, taking 500 mu L of E.coli bacterial liquid collected in step 2.1 in a 1.5mL centrifuge tube containing 200 mu L of streptomycete spore suspension, mixing uniformly, taking 200 mu L of mixed liquid, uniformly coating on a MS flat plate, pouring the MS flat plate into a 30 ℃ incubator for culture, adding 1mL of antibiotic premix (1 mg/mL of apramycin and 1mg/mL of nalidixic acid concentration) after 18 hours, and continuously pouring the MS flat plate into 30 ℃ until the binder grows out (about 5 days). At this time, a binder was obtained which was successfully edited.

Example 3 evaluation of Gene editing efficiency

Picking up the single clone in the step 2.2, streaking and inoculating to ISP2 solid culture medium with 25 mug/mL apramycin and 0.5 mug/mL thiostrepton, inverting, culturing at 30 ℃ to visible thalli at constant temperature, picking up a small quantity of thalli to a PCR tube containing 20 mug DMSO, placing at 100 ℃ for 15 minutes, refrigerating at-20 ℃ for 30 minutes, repeating the steps twice to fully lyse cells to obtain cell lysate, and then adopting a Novox 2X PHANTA FLASH MASTER Mix (product number: P510-01) kit for PCR amplification of target site fragments, wherein the design of primers is shown in table 1, the PCR reaction system is shown in table 2, and the PCR reaction procedure is shown in table 3.

TABLE 1 PCR amplification primers targeting SCO5087 (actI)

Numbering device	Primer(s)	Sequence (5 '-3')
			SEQ ID NO.9	SCO5087-LH-F	atgattccggaactccggt
SEQ ID NO.10	SCO5087-R	accacagcttgcggaact

TABLE 2PCR reaction System

System of	15μL
		ddH₂O	5.25μL
Forward primer (10. Mu.M, SCO 5087-LH-F)	0.75μL
		Reverse primer (10. Mu.M, SCO 5087-R)	0.75μL
2×Phanta Flash Master Mix	7.50μL
		Cell lysate (containing DMSO)	0.75μL

Table 3 PCR reaction procedure

The PCR amplification products were subjected to 1% agarose gel electrophoresis, and the partial results are shown in FIG. 3, wherein 1-21 in FIG. 3 represent different single clones randomly picked after E.coli containing HpTnpB-reRNA-ZJ plasmid was combined with S.coelicolor A3 (2) for transfer. If the target gene SCO5087 (actI) was successfully knocked out by plasmids HpTnpB-reRNA-ZH or HpTnpB-reRNA-ZJ containing homologous recombination repair templates, the theoretical PCR band size should be 1329 bp (FIG. 2). The randomly picked colonies growing in the resistant solid screening culture medium are provided with PCR products by PCR and have the sizes consistent with theory, which shows that HpTnpB-reRNA-ZH and HpTnpB-reRNA-ZJ plasmids can successfully knock out a target gene SCO5087 (actI) in a Streptomyces mode strain S.ceruicolor A3 (2), the knock-out efficiency of a plasmid system (HpTnpB-reRNA-ZH) on the target gene is 69.5% under the action of non-optimized guide RNA, and the knock-out efficiency of the plasmid system (HpTnpB-reRNA-ZJ) is up to 100% under the action of optimized guide RNA (figure 4), which shows that the knock-out efficiency of the plasmid system is remarkably improved after the guide RNA sequence is optimized. Homologous recombination is one of the main methods for achieving gene knockout and gene knock-in, and the homologous sequences on the target genome are subjected to recombination reaction in vivo through exogenous DNA fragments (namely homologous arms on the upper and downstream of the target gene), so that the target gene is knocked out or replaced (namely, the exogenous gene is inserted into the position of the target gene, so that the gene knock-in is achieved). The mechanism between gene knockout and homologous knockout is based on homologous recombination, and the supermini gene editor is proved to be applicable to knockout/into other target genes.

The PCR product was then recovered by purification using a Renzan plasmid purification kit (cat# DC 201) and sent to Sanger sequencing analysis, inc. of Jin Weizhi Biotechnology, su. The sequencing results further confirm the results of the gel electrophoresis detection (FIG. 5). The above results fully demonstrate that the novel supermini gene editor HpTnpB-reRNA-ZJ obtained by the present invention can effectively mediate genome editing of Streptomyces.

Example 4 various media according to the respective examples

LB medium 10g tryptone, 5g yeast extract and 10g sodium chloride were weighed, dissolved in 1L ddH ₂ O, sterilized at 115℃for 30 minutes and stored at room temperature for use. If a solid culture medium is prepared, 2% of agar powder is additionally added;

MS culture medium, 10g soybean cake powder, 10g tryptone and 10g agar powder are weighed, dissolved in 500mL tap water and sterilized at 115 ℃ for 30 minutes;

ISP2 culture medium, namely weighing 10g of malt extract, 4g of yeast extract and 4g of glucose, dissolving in 1L of ddH ₂ O, regulating the pH to 7.4,115 ℃ for sterilization for 30 minutes, storing at 4 ℃ for standby, and adding 2% of agar powder if a solid culture medium is configured;

2 XYT medium 16g tryptone, 10g malt extract, 5g sodium chloride are weighed, dissolved in 1L ddH ₂ O, pH is adjusted to 7.0,115 ℃ and sterilized for 30 minutes, and stored at 4 ℃ for later use.

The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.

Claims

1. A guide RNA for guiding TnpB protein to move to a target sequence, characterized in that the guide RNA comprises an RNA backbone, a gene targeting segment, and a gene sequence of a hepatitis D virus ribozyme; the nucleotide sequence of the RNA backbone is SEQ ID NO.3 or SEQ ID NO.6.

2. The guide RNA according to claim 1, wherein the gene targeting segment is located at the 3' end of the RNA backbone and is a nucleic acid fragment with a length of 12-40 bp following the TAM sequence 5'TTGAT on the target gene.

3. A TnpB-mediated Streptomyces mini-gene editing system, characterized in that the system comprises a TnpB nuclease that can be expressed in Streptomyces and the guide RNA according to claim 1 or 2.

4. The Streptomyces mini gene editing system according to claim 3, characterized in that the amino acid sequence of the TnpB nuclease is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

5. A recombinant expression plasmid vector, characterized in that it is used to express the Streptomyces mini gene editing system as described in claim 3 or 4.

6. A method for constructing a Streptomyces mini gene editing system according to claim 3 or 4, characterized in that the method comprises constructing a gene knockout/knock-in plasmid containing an apramycin resistance selection marker; the gene knockout/knock-in plasmid is based on the Escherichia coli-Streptomyces shuttle plasmid, and the TnpB nuclease and its guide RNA that can be expressed in Streptomyces and the gene knockout/knock-in cassette are inserted.

7. The construction method according to claim 6 is characterized in that the gene knockout cassette includes the following core elements in sequence according to the direction of the gene editing target gene: an upstream homologous arm of the target gene and a downstream homologous arm of the target gene; the upstream and downstream homologous sequences of the target gene are used to provide a homologous recombination template when gene editing occurs.

8. The construction method according to claim 6 is characterized in that the gene knock-in cassette includes the following core elements in sequence according to the direction of the gene editing target gene: an upstream homology arm of the target gene, an inserted gene, and a downstream homology arm of the target gene.

9. An application of the Streptomyces mini-gene editing system as described in claim 3 or 4 in the precise editing of the Streptomyces genome based on double-strand break and directed repair.

10. A method for gene editing a target gene in a recipient bacterium Streptomyces using the Streptomyces mini gene editing system according to claim 3 or 4, characterized in that the method comprises the following steps:

S1. Under non-inducing conditions, the gene knockout/in plasmid constructed by the method of claim 6 is transformed into the target host, and the gene knockout/in is achieved by homologous exchange of the homologous arm sequence of the gene knockout/in cassette with the upstream and downstream homologous sequences of the target gene. Transformants carrying the gene knockout/in plasmid are obtained by screening for antibiotics associated with the plasmid resistance;

S2. The transformant is streaked onto a culture medium containing a plasmid resistance antibiotic and a promoter inducer, and cultured at a constant temperature until a single colony is visible;

S3. Randomly select single clones from step S2 and verify by colony PCR to obtain a traceless gene knockout/in strain.