CN120683078A - A TnpB gene editing system suitable for Streptomyces and its construction and application - Google Patents

Abstract

The present invention discloses a TnpB gene editing system suitable for Streptomyces, and its construction and application; the system includes an engineered TnpB nuclease and a reRNA guide cassette, wherein the engineered TnpB nuclease has an amino acid difference relative to the amino acid sequence shown in SEQ ID NO.1. Furthermore, the amino acid difference is located at one of positions 188, 217, 125, 179, 388, 27, 25, 8, 96, 240, 208, 279, 254, 110, 267, 286, 186, 356, 162, 210, 332, 385, 9, 200, 111, 333, and 57. The engineered TnpB nuclease and its effector protein of the present invention have higher activity and show better gene editing efficiency in Streptomyces; TnpB mutants (such as D333V, S57R, etc.) have an editing efficiency of nearly 100% for the endogenous Streptomyces gene SCO5087.

Description

TnpB gene editing system suitable for streptomycete, construction and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, relates to a TnpB gene editing system suitable for streptomyces, and construction and application thereof, and in particular relates to an engineering TnpB nuclease, tnpB gene editing system and a gene editing method suitable for streptomyces. The engineered TnpB nuclease comprises a mutation based on a reference TnpB nuclease. The engineering TnpB and TnpB gene editing system can realize efficient and accurate gene editing in streptomycete, and compared with a TnpB gene editing system which is not engineering, the engineering TnpB nuclease and TnpB gene editing system can greatly improve the editing efficiency of target genes.

Background

Streptomyces is the biggest genus in actinomycota, and has the potential of encoding new compounds in a large amount due to the fact that secondary metabolites with abundant structures, wide activity and great application value can be produced, and the streptomyces has wide application prospects in the fields of medicine and health, food, industry and agriculture and the like. However, due to the characteristics of streptomycete, such as high GC content in genome, linear chromosome, most of biosynthesis gene clusters of secondary metabolites under experimental conditions, and the like, systematic metabolic engineering by a traditional homologous double-exchange mode is greatly limited to develop new valuable secondary metabolites or improve the yield of the secondary metabolites with known activities. The efficient bottom-up excavation of the existing secondary metabolites from the genome by utilizing the synthetic biology is a main stream idea of the development of the existing secondary metabolites, and the possession of a powerful genetic operating system is a basis for the development and improvement of the existing secondary metabolites from streptomyces.

However, streptomyces has a large genome (8-10 Mb) and a high GC content (generally, GC content is more than 70%), and genetic manipulation such as gene editing is difficult to accomplish on Streptomyces as compared with other microorganisms, and genetic manipulation means are limited. With the development of CRISPR-Cas9 technology and the application thereof in streptomyces, the problems of time consuming and low efficiency of streptomyces gene editing are greatly alleviated, the bottleneck of lack of efficient genetic operation in the field of streptomyces is effectively broken, and the method becomes a main gene editing tool applicable to streptomyces at present. Although CRISPR-Cas9 series gene editing tools play an important role in streptomycete gene editing, there are a number of problems to be improved. Such as CRISPR-Cas9 is too bulky, affecting plasmid construction and delivery efficiency, and PAM sequences with high GC lead to potential high Off-target effects (Off-TARGET EFFECTS) and other problems.

In order to break through the application limitation caused by large protein volume such as Cas9, researchers change the key elements of the CRISPR-Cas system through system optimization to overcome the defects, such as using a series of measures such as orthologous enzyme of Cas9, modifying Cas9, optimizing guide RNA and the like, on the other hand, the application of novel gene editing technology such as base editing, PRIME EDITING and the like is continuously explored, and in addition, the researchers continuously excavate and develop compact CRISPR proteins to develop a novel CRISPR system. TnpB is a recently discovered programmable nuclease of only about 1/3 (about 400 amino acids) of Cas9 protein, which can cleave DNA sequences near the 5' end of TTGAT under the guidance of long non-coding RNAs, and is of interest to researchers at home and abroad. To date, tnpB has been successfully used to edit endogenous genes of multiple species, but in general, tnpB has a problem of low editing efficiency compared with Cas9, as compared with other reported compact gene editing tools, and more importantly, the existing small gene editor has not been reported to edit in streptomyces.

Disclosure of Invention

The invention aims to provide a TnpB gene editing system suitable for streptomycete, and construction and application thereof. Aiming at the current situation that a mini gene editing tool is not available in streptomyces and the problem that the existing mini gene editing tool is generally low in editing efficiency, a streptomyces mini gene editing system between TnpB is constructed. On the basis, through deep analysis and rational design of TnpB-DNA-RNA three-dimensional crystal structure, tnpB nuclease is optimized and modified, a streptomycete efficient mini gene editing system between TnpB nucleases is developed, and accurate and efficient editing of genome in streptomycete is realized, so that a streptomycete gene operation tool library is enriched, the study of gene function of a streptomycete silent gene cluster is promoted, and a powerful enabling tool is provided for accurate modification of metabolic pathways and intelligent biological manufacturing.

In order to overcome the problem of low editing efficiency of the existing mini gene editor, the invention further enriches a streptomycete gene editing tool library, builds a mini streptomycete gene editing system based on TnpB based on TnpB nuclease (only about 400 amino acids), and develops a streptomycete efficient gene editing system based on engineering TnpB nuclease and a gene editing method and flow corresponding to the system by analyzing and rationally designing a TnpB-DNA-RNA three-dimensional crystal structure and engineering TnpB nuclease. The invention aims at realizing the following technical scheme:

In a first aspect, the invention provides an engineered TnpB nuclease having an amino acid difference relative to the amino acid sequence shown in an unmodified TnpB nuclease (as set forth in SEQ ID NO. 1).

As one embodiment of the present invention, the amino acid difference is located at one of 188 th, 217 th, 125 th, 179 th, 388 th, 27 th, 25 th, 8 th, 240 th, 208 th, 96 th, 279 th, 254 th, 110 th, 267 th, 286 th, 186 th, 356 th, 162 th, 210 th, 332 th, 385 th, 9 th, 200 th, 111 th, 333 th, 57 th.

As one embodiment of the present invention, the amino acid difference is preferably located at one of 356 th, 162 th, 210 th, 332 th, 385 th, 9 th, 200 th, 111 th, 333 th, 57 th.

In some preferred embodiments, the engineered TnpB nuclease has an amino acid residue A substituted with V at position 188, an amino acid residue S substituted with K at position 217, an amino acid residue N substituted with G at position 125, an amino acid residue Y substituted with P at position 179, an amino acid residue Y substituted with A at position 388, an amino acid residue S substituted with C at position 27, an amino acid residue L substituted with F at position 25, an amino acid residue V substituted with K at position 8, an amino acid residue G substituted with R at position 240, an amino acid residue H substituted with K at position 208, an amino acid residue T substituted with R at position 96, an amino acid residue H substituted with T at position 279, an amino acid residue V substituted with S at position 254, an amino acid residue R substituted with K at position 110, an amino acid residue S substituted with R at position 267, an amino acid residue R substituted with L at position 286, an amino acid residue F substituted with K at position 186, an amino acid residue E substituted with F at position 356, an amino acid residue E substituted with F at position 8, an amino acid residue V substituted with K at position 240, an amino acid residue T substituted with F at position 37, an amino acid residue T substituted with F at position 75, an amino acid residue F substituted with F at position 57, an amino acid residue V substituted with F at position 75, an amino acid residue C substituted with F at position amino acid residue L substituted with F at position 267, an amino acid residue V substituted with F preferred.

In some preferred embodiments, the engineered TnpB nuclease has an A substitution at amino acid residue E at 356, an N substitution at amino acid residue I at 162, an N substitution at amino acid residue Q at 210, an S substitution at amino acid residue H at 332, a C substitution at amino acid residue H at 385, a Y or F substitution at amino acid residue V at 9, preferably F substitution at 200, an L substitution at amino acid residue V at 111, an S substitution at amino acid residue K at 333, a V substitution at amino acid residue S at 57, and an R substitution at amino acid residue S at 57.

In a second aspect, the invention provides a system for achieving efficient minigene editing in streptomyces, comprising the engineered TnpB nuclease and an RNA-guided cassette thereof.

As one embodiment of the invention, the reRNA guide box sequentially comprises the following core elements of a guide RNA base sequence of TnpB protein, a gene targeting segment and a Hepatitis Delta Virus (HDV) ribozyme according to the direction of a gene editing target gene, and is used for guiding TnpB protein to move to target sequence DNA.

As one embodiment of the invention, the nucleotide sequence of the guide RNA of TnpB protein is shown as SEQ ID NO. 3.

As one embodiment of the present invention, the gene targeting segment, which is located at the 3 '-end of the guide RNA base sequence, is a nucleic acid fragment having a length of 12-40bp after the TAM sequence 5' TTGAT on the target gene.

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

In a fourth aspect, the invention provides a construction method of the streptomyces minigene editing system, which comprises the step of constructing a gene editing plasmid containing an apramycin resistance screening marker, wherein the gene editing plasmid is based on an escherichia coli-streptomyces shuttle plasmid, and the engineering TnpB nuclease and a reRNA guide box thereof are inserted.

In a fifth aspect, the present invention provides a Streptomyces minigenome editing system as described above for use in a repair system based on double strand breaks and Streptomyces itself.

In a sixth aspect, the present invention provides a method for gene editing suitable for Streptomyces, the method comprising transferring the above gene editing system into a host of Streptomyces of interest under non-induced conditions, deleting or knocking in or replacing genes near the target site of the host, causing frame shift mutation, and further inactivating the target gene.

As one embodiment of the present invention, the gene editing method comprises the steps of:

S1, under a non-induction condition, transforming the gene editing plasmid constructed by the method into a target host, and connecting the homologous tail ends of streptomycete to generate gene deletion, insertion or replacement near a target site to cause frame shift mutation so as to inactivate a target gene;

S2, streaking and inoculating the transformant to a culture medium containing plasmid resistance antibiotics and promoter inducers (such as thiostrepton), and culturing at constant temperature until the transformant is visible monoclonal;

S3, randomly selecting the monoclonal in the step S2, and verifying colony PCR to obtain the target gene mutant strain.

Compared with the prior art, the invention has the following beneficial effects:

Compared with the conventional TnpB gene editing tool, the engineering TnpB nuclease and the effector protein thereof have higher activity, and the engineering TnpB nuclease has more excellent gene editing efficiency in streptomycete bodies, for example, the editing efficiency of a plurality of TnpB (such as D333V, S57R and the like) mutants in the embodiment of the invention on the streptomycete endogenous gene SCO5087 is close to 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 an engineered TnpB nuclease editing efficiency, note that WT is an unengineered TnpB nuclease, and the dotted bar graph shows editing efficiency 2-fold and above that of the unengineered TnpB nuclease;

FIG. 2 is a diagram showing the result of electrophoresis (TnpB (S57V));

FIG. 3 is a diagram showing the sequencing result (TnpB (S57V));

FIG. 4 is a graphical representation of the results of the inactivation of target genes by engineered TnpB nuclease (TnpB (S57V)).

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.

Example 1 engineering TnpB nucleases

1.1 Plasmid design and construction

According to the codon preference of streptomyces, the TnpB nuclease from Deinococcus radiodurans ISDra is subjected to codon optimization, and synthesized by Kirschner Biotechnology Inc., the amino acid sequence of TnpB is shown as SEQ ID NO.1, the gene sequence is shown as SEQ ID NO.2, and the DNA similarity with the Deinococcus radiodurans ISDra TnpB nuclease from TnpB is 79.74%.

A reRNA guide box for guiding a wild-type TnpB nuclease to play a double-strand cutting function near an endogenous target site of streptomycete sequentially comprises a TnpB protein guide RNA base sequence (SEQ ID NO. 3), a gene targeting segment and a Hepatitis Delta Virus (HDV) ribozyme (SEQ ID NO. 4) according to the direction from 5 'to 3' end. The gene targeting segment is positioned at the 3 'end of the RNA framework and is a nucleic acid fragment with the length of 12-40bp after a TAM sequence (5' TTGAT) on a target gene. According to the embodiment of the invention, a key gene SCO5087 (actI) for actinorhodin synthesis of streptomycete model strain Streptomycin coelicolor A (2) (genome sequence number: geneBank: GCA_ 008931305.1) is selected as a cell endogenous gene target, and the base sequence of a gene targeting segment in a reRNA guide box corresponding to the target is 5' -GTAGTCGATGTCCGTCGCGT. The reRNA guide cassette used in the examples was synthesized by the company Kirsry Biotech Co.

The synthesized wild-type TnpB and its reRNA guide cassette were substituted for the Cas9 and sgRNA fragments on the Streptomyces coli shuttle plasmid vector pCRISPR-Cas9 (https:// doi.org/10.1021/acslynbio.5b00038), respectively, to obtain plasmid pTnpB-ZH.

According to the Deinococcus radiodurans ISDra source TnpB nuclease, guide RNA and substrate DNA combined crystal structure, 80 single-point mutations of TnpB are designed (shown in table 1), pTnpB-ZH is used as a template, mutation is introduced by inverse PCR (shown in table 2) to realize site-directed mutation of TnpB, and primers are shown in table 3, wherein 2X MegaPfu Premix (with dye) is purchased from ipecac harbor organisms (product number: 21809), and then the inverse PCR product is purified (a kit is purchased from Nanjinovone biotechnology Co., ltd.); Gel DNA Extraction Mini Kit, DC 301), and then transferring the purified PCR product into competent cell E.coli DH5 alpha, and utilizing its own DNA recombination repair system to implement in vivo cyclization of linear DNA so as to obtain TnpB optimized plasmid system.

The constructed plasmids were all Sanger sequenced to ensure complete correctness.

Table 1TnpB nuclease single point mutation sequences

TABLE 2 inverse PCR reaction System

TABLE 3 reverse PCR primers and annealing temperatures

Example 2 application of optimized Streptomyces after Mini-editing System

2.1 Conversion

The method comprises transferring TnpB optimized plasmids into self-made competent cells E.coli ET12567/pUZ8002 (https:// doi.org/10.1016/0378-1119 (92) 90549-5), respectively, adding 200ng TnpB optimized plasmids into 100 μl of thawed E.coli ET12567/pUZ8002 competent cells, mixing, standing on ice for 30 min at 42 ℃ for 45 seconds, immediately placing on ice for 2 min, adding 400 μl of antibiotic-free LB liquid medium, culturing at 37 ℃ for 1 hr at 200rpm, then coating 100 μl on LB solid plates containing kanamycin (25 μg/mL), chloramphenicol (12.5 μg/mL) and amphotericin (50 μg/mL), culturing at 37 ℃ and then taking 20mL of LB solid plates containing 25 μg/mL, chloramphenicol (12.5 μg/mL) and amphotericin (50 μg/mL), adding 20mL of LB solid plates containing kanamycin (34 μg/mL), centrifuging liquid medium at 500 rpm for 2 min, and culturing at 50 μg/50 mg, and standing overnight, and collecting the liquid medium after centrifugation.

2.2 Binding transfer and resistance screening

Under non-induction conditions, the plasmid in step 2.1 was transferred to S.coelicolor A3 (2) by combination transfer, and the specific operation method was as follows, taking Streptomyces spores collected in advance, centrifuging (5000 rpm,5 min), discarding the supernatant, then resuspending in 2mL of 2 XYT liquid medium, heat-shocking for 10 min at 50 ℃ and then pre-germinating for 30 min in a 30 ℃ shaker, taking 500. Mu.L of E.coli bacterial liquid collected in step 2.1 and 200. Mu.L of Streptomyces spore suspension in a 1.5mL centrifuge tube, mixing well, taking 100. Mu.L of mixed liquid, coating on MS plates, pouring the MS plates in a 30 ℃ incubator for culturing, covering the surface of the medium with apramycin (1 mg/mL) and nalidixic acid (1 mg/mL) after 18 hours, continuing pouring the MS plates in 30 ℃ until the binder grows (about 5 days) after surface drying. At this time, a successfully edited binder is obtained.

Example 3 evaluation of Gene editing efficiency of engineering TnpB System

Randomly picking the resistant binder in the step 2.2, streaking and inoculating the binder on ISP2 solid medium with plasmid resistance antibiotics (50 ng/mL apramycin) and thiostrepton (0.5 mug/mL), inverting, culturing at 30 ℃ until the bacterial cells are visible, picking a small amount of bacterial cells to a PCR tube containing 20 mug of DMSO, lysing the cells in a metal bath (100 ℃ for 15 minutes) to obtain a cell lysate, and evaluating the editing efficiency of the optimized TnpB system by adopting a colony PCR mode system, wherein the specific operation is as follows, PCR amplification of target site fragments by adopting a kit (purchased from Nannofa Biotechnology Co., ltd., product number: P222-01) is shown in table 4, a PCR reaction system is shown in table 5, and a PCR reaction program is shown in table 6.

TABLE 4 PCR amplification primers targeting SCO5087 (actI)

Numbering device	Primer(s)	Sequence (5 '-3')
			SEQ ID NO.5	SCO5087-LH-F	atgattccggaactccggt
SEQ ID NO.6	SCO5087-R	accacagcttgcggaact

TABLE 5PCR reaction System

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

TABLE 6PCR reaction procedure

In this example, 2% agarose gel electrophoresis detection or Sanger sequencing or whole genome resequencing was used in combination to determine whether randomly selected binders were edited. TnpB is matched with the defective non-homologous end connection of streptomycete, so that the editing of target genes can be realized, such as gene knockout, insertion, replacement and the like. If successfully edited, the PCR amplification product is subjected to 2% agarose gel electrophoresis, the band size is different from that of the wild type, samples which cannot be judged by gel electrophoresis are purified and recovered by using a Renzan plasmid purification kit (product number: DC 201) to judge whether the edited samples are sent to Sanger sequencing analysis by Jin Weizhi Biotechnology Inc. in Suzhou, and samples without amplification products after PCR are collected and sent to the biological (Shanghai) stock Inc. for whole genome resequencing, so that the gene editing condition is further evaluated. The results show (fig. 1-3), targeting the target gene SCO5087 (actI) in model strain s.ceruicolor A3 (2), single point mutation after TnpB locus (A188V、S217K,N125G,Y179P,Y388A,S27C,L25F,V8K,G240R,H208K,T96R,H279T,V254S,R110K,S267R,R286L,F186K,E356A,I162N,Q210N,H332S,H385C,V9Y,V200L,K111S,V9F,D333V,S57R) exhibits high efficiency of gene editing, has at least 2-fold enhancement of gene editing capacity, wherein TnpB nuclease comprises one of 356 th, 162 th, 210 th, 332 th, 385 th, 200 th, 111 th, 9 th, 333 th and 57 th amino acids after mutation, and the editing efficiency is improved 3.0-4.3-fold compared with wild type TnpB system.

Example 4 inactivation of target Gene Using engineered TnpB enzymes

Under non-induced conditions, tnpB-optimized plasmid system (S57R) was transferred into S.coelicolor A3 (2) by means of a binding transfer, as in step 2.2. After about 5 days, resistant binders were randomly picked, streaked onto ISP2 solid medium with plasmid resistant antibiotics (50 ng/mL apramycin) and thiostrepton (0.5. Mu.g/mL), inverted, cultured at 30℃and observed for pigment secretion. According to the embodiment of the invention, a key gene SCO5087 (actI) synthesized by actinorhodin is taken as a cell endogenous gene target, and after the target gene is mutated, S.coelicolor A3 (2) cannot secrete blue pigment. The detection of target genes by colony PCR or whole genome resequencing is used to determine whether randomly selected binders are edited, as described in example 3. The result display of the inactivation of the target gene by the engineered TnpB nuclease (TnpB (S57V)) is shown in FIG. 4, and the mutant strain of the target gene is cultivated in the resistance plate for 2 weeks, and no blue pigment secretion is seen, which further shows that the repair mode of the engineering TnpB system combined with the non-homologous end joining (NHEJ) of streptomycete has gene deletion or gene knock-in and replacement near the target site, so that the target gene frame shift mutation is caused, and the target gene is inactivated.

Example 5 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 (10)

1. An engineered TnpB nuclease for use in a streptomyces minigene editing system, wherein the engineered TnpB nuclease has an amino acid difference relative to a TnpB nuclease having an amino acid sequence as set forth in SEQ ID No. 1.

2. The engineered TnpB nuclease of claim 1, wherein the amino acid difference is at one of position 188, position 217, position 125, position 179, position 388, position 27, position 25, position 8, position 240, position 208, position 96, position 279, position 254, position 110, position 267, position 286, position 186, position 356, position 162, position 210, position 332, position 385, position 9, position 200, position 111, position 333, position 57 of the amino acid sequence as set forth in SEQ ID No. 1.

3. The engineered TnpB nuclease of claim 1 or 2, wherein the engineered TnpB nuclease has one of the following mutations in the amino acid sequence set forth in SEQ ID No.1, ：A188V、S217K、N125G、Y179P、Y388A、S27C、L25F、V8K、G240R、H208K、T96R、H279T、V254S、R110K、S267R、R286L、F186K、E356A、I162N、Q210N、H332S、H385C、V9Y or V9F, V200L, K111S, D333V, S R.

4. A streptomyces minigene editing system comprising the engineered TnpB nuclease and reRNA leader cassette of any one of claims 1-3.

5. The streptomycete minigene editing system of claim 4, wherein the reRNA guide box comprises a guide RNA base sequence of TnpB protein, a gene targeting segment, and a gene sequence of hepatitis delta virus ribozyme.

6. The Streptomyces minigene editing system according to claim 5, wherein the gene targeting segment is located at the 3 'end of the guide RNA base sequence and is a nucleic acid fragment having a length of 12 to 40bp after the TAM sequence 5' TTGAT on the target gene.

7. A method for constructing a Streptomyces minigene editing system according to any one of claims 4 to 6, comprising constructing a gene editing plasmid containing an apramycin resistance selection marker, wherein the gene editing plasmid is based on an E.coli-Streptomyces shuttle plasmid, and inserting the engineered TnpB nuclease and its reRNA guide cassette.

8. A recombinant expression plasmid vector for expressing the streptomyces minigene editing system of any one of claims 4-6.

9. Use of the streptomycete minigene editing system according to any one of claims 4-6 for editing the genome of streptomycete based on double strand breaks and the streptomycete self repair system.

10. A method for gene editing suitable for streptomyces, characterized in that the streptomyces minigene editing system according to any one of claims 6-8 is transferred into a host of streptomyces of interest by combining transfer under non-induced conditions, so that gene deletion or gene knock-in and substitution occurs near the target site of the host, and frame shift mutation is caused, thereby inactivating the target gene.