CN120683080A - An engineered TnpB nuclease and its editing system and application - Google Patents

Abstract

The present invention discloses an engineered TnpB nuclease and its editing system and application. The engineered TnpB nuclease of the present invention undergoes site-directed mutagenesis of amino acid sites relative to the wild-type TnpB nuclease whose amino acid sequence is shown in SEQ ID NO.1, and the amino acid sequence of the engineered TnpB nuclease is shown in SEQ ID NO.3. The present invention also discloses a guide RNA, a Streptomyces mini-gene editing system comprising the engineered TnpB nuclease and the guide RNA, a recombinant expression plasmid expressing the editing system, a method for constructing the editing system, and the application of the editing system in mediating the Streptomyces mini-gene editing system. The engineered TnpB nuclease of the present invention, combined with the Streptomyces mini-gene editing system formed by the guide RNA, can perform efficient gene editing of Streptomyces near the 5'-TTGAT sequence, greatly improving the editing efficiency.

Description

Engineered TnpB nuclease and editing system and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an engineered TnpB nuclease and an editing system and application thereof.

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 genetic manipulation such as gene editing is difficult and genetic manipulation is limited as compared with other microorganisms.

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.

Disclosure of Invention

Aiming at the current situation that a mini gene editing tool is not available in streptomyces, the invention deeply analyzes a small-volume programmable TnpB nuclease to realize a core element for gene editing and an action mechanism thereof, systematically digs potential TnpB nuclease, realizes the cutting of target double chains through engineering transformation, systematically develops a high-efficiency mini gene editing tool suitable for the streptomyces, and further realizes the high-efficiency and accurate editing of the genome of the streptomyces. The invention aims to provide an engineering TnpB nuclease and an editing system and application thereof.

The aim of the invention is realized by the following technical scheme:

In a first aspect, the invention provides an engineered TnpB nuclease suitable for use in Streptomyces, the engineered TnpB nuclease having an amino acid site mutation comprising position 41, position 42, position 44, position 45, position 46, position 50, position 51, position 53, position 54, position 56, position 57, position 58, position 64, position 65, position 67, position 69, position 72, position 81, position 84, position 85, position 88, position 91, position 97, position 99 relative to a wild-type TnpB nuclease having the amino acid sequence shown in SEQ ID NO. 1.

As some embodiments of the invention, the amino acid site mutation comprises ：E41A、S42A、R44K、Q45E、D46S、M50L、I51T、A53G、A54Q、D56S、K57S、A58E、R64Q、E65A、G67E、A69S、Q81N、R84K、D85N、R88T、Q91K、A97V,K99Q; the amino acid sequence of the engineered TnpB nuclease is shown in SEQ ID NO. 3.

As some specific embodiments of the invention, the nucleotide sequence of the engineered TnpB nuclease is shown as SEQ ID NO. 4.

As some specific embodiments of the invention, the wild-type TnpB nuclease is TnpB nuclease which is expressed in Streptomyces through codon optimization, and the nucleotide sequence of the TnpB nuclease is shown as SEQ ID NO. 2.

In a second aspect, the invention provides an engineered TnpB nuclease-mediated Streptomyces minigene editing system comprising an engineered TnpB nuclease as described in any one of the above, and a guide RNA.

As some embodiments of the invention, the guide RNA comprises an RNA backbone, a gene targeting segment, and a Hepatitis Delta Virus (HDV) ribozyme for directing movement of the engineered TnpB nuclease toward a target sequence.

As some specific embodiments of the invention, the RNA skeleton is a segment of RNA nucleotide sequence which is reasonably designed by the invention, the nucleotide sequence is shown as SEQ ID NO.5, the gene targeting segment is positioned at the 3 'end of the RNA skeleton and is a nucleic acid segment with the length of 12-40bp after a TAM sequence (5' -TTGAT) on a target gene, and the nucleotide sequence of the Hepatitis Delta Virus (HDV) ribozyme is shown as SEQ ID NO.7 and is used for stabilizing the structure of the RNA skeleton-gene targeting segment.

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

In a fourth aspect, the invention provides a method for constructing a Streptomyces minigene editing system, comprising constructing a gene editing plasmid containing an apramycin resistance selection marker, wherein the gene editing plasmid is constructed by inserting the engineering TnpB nuclease and the guide RNA on the basis of an E.coli-Streptomyces shuttle plasmid.

In a fifth aspect, the present invention provides the use of a Streptomyces minigene editing system for mediating Streptomyces gene editing.

As some specific embodiments of the present invention, the method for gene editing of Streptomyces using the Streptomyces minigene editing system comprises the steps of:

s1, under the non-induction condition, transforming a gene editing plasmid into a target host, editing a target gene through a DNA repair mechanism of the streptomycete in vivo, and screening antibiotics with plasmid related resistance to obtain a transformant carrying the gene knockout/plasmid entry;

S2, streaking and inoculating the transformant in the step S1 to a culture medium plate containing plasmid resistance antibiotics and promoter inducers, and culturing at constant temperature until the single clone is visible;

S3, randomly selecting the monoclonal in the step S2, and performing colony PCR verification to obtain the edited strain.

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

(1) Compared with the corresponding wild type TnpB, the novel engineered TnpB nuclease provided by the invention can recognize the 5' -TTGAT sequence and realize efficient editing of the sequence nearby in streptomycete under the action of the guide RNA of the physical design after being subjected to engineering transformation, and has great popularization and application values;

(2) The invention provides a novel streptomycete mini gene editing tool, a construction method and application thereof, wherein in streptomycete, the possibility of editing near TTGAT by a wild type TnpB is extremely low (about 8%), but the gene editing efficiency under the action of TnpB after engineering modification is remarkably improved and can reach 41%.

Drawings

Other features, objects and advantages of the present 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 shows plasmid maps pSTAGE-TnpB and pSTAGE-eTnpB for omega RNA constructed in example 1, wherein the left panel shows plasmid maps pSTAGE-TnpB for omega RNA and the right panel shows plasmid maps pSTAGE-eTnpB for omega RNA;

FIG. 2 is a graph showing the results of PCR electrophoresis of randomly selected portions of a single clone from Streptomyces endogenous gene editing using pSTAGE-TnpB x/ωRNA;

FIG. 3 is a graph showing the results of PCR electrophoresis of randomly selected portions of a monoclonal antibody obtained by endogenous gene editing of Streptomyces with pSTAGE-eTnpB. Omega. RNA;

FIG. 4 is a graph comparing editing efficiency of endogenous gene editing of Streptomyces using pSTAGE-TnpB and pSTAGE-eTnpB. Omega. RNA;

FIG. 5 is a graph of Sanger sequencing results of a portion of a monoclonal strain of Streptomyces endogenous gene editing using pSTAGE-eTnpB. Omega. RNA.

Detailed Description

The present invention will be described in detail with reference to specific 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 variations and modifications could 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 Streptomyces coelicolorA (2) as a target strain, and the genome sequence number of the strain is GeneBank:GCA_008931305.1. The embodiment of the invention takes the key gene SCO5087 (actI) synthesized by actinorhodin in the strain as an endogenous gene target spot, takes the editing SCO5087 as a target, and tests the editing efficiency of the invention.

EXAMPLE 1 construction of Streptomyces efficient Mini gene editing tool

1.1 Plasmid design and construction

According to the codon preference of streptomyces, the TnpB nuclease from Deinococcus radiodurans R sources is subjected to codon optimization, and is named as wild TnpB nuclease, the amino acid sequence of the nuclease is shown as SEQ ID NO.1, and the gene sequence after the codon optimization is shown as SEQ ID NO. 2.

Site directed mutagenesis ：E41A、S42A、R44K、Q45E、D46S、M50L、I51T、A53G、A54Q、D56S、K57S、A58E、R64Q、E65A、G67E、A69S、Q81N、R84K、D85N、R88T、Q91K、A97V,K99Q, was performed on wild-type TnpB nuclease to obtain an engineered TnpB nuclease (eTnpB). The amino acid sequence of the engineered TnpB x nuclease (eTnpB x) is shown as SEQ ID NO.3, and the gene sequence is shown as SEQ ID NO. 4.

The guide RNA is named omega RNA, and sequentially comprises the following core elements of an RNA framework (the sequence is shown as SEQ ID NO. 5), a gene targeting segment (the sequence is shown as SEQ ID NO.6 when SCO5087 is taken as a target point) and a Hepatitis Delta Virus (HDV) ribozyme (the sequence is shown as SEQ ID NO. 7) according to the direction from 5 'to 3'.

The above gene fragments were synthesized by Kirschner Biotech Co., ltd.

The synthesized wild-type TnpB and omega RNA gene fragments described above were substituted for Cas9 and sgRNA fragments on the streptomycete-e.coli shuttle plasmid vector pCRISPR-Cas9 (https:// doi. Org/10.1021/acslynbio. 5b 00038), respectively, to obtain plasmids pSTAGE-TnpB/omega RNA, as shown in the left panel of fig. 1.

The synthetic engineered eTnpB and omega RNA gene fragments described above were substituted for Cas9 and sgRNA fragments on the streptomycete-escherichia coli shuttle plasmid vector pCRISPR-Cas9 (https:// doi. Org/10.1021/acslynbio. 5b 00038), respectively, to obtain plasmids pSTAGE-eTnpB/omega RNA, as shown in the right panel of fig. 1.

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

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

2.1 Conversion

The plasmids pSTAGE-TnpB and pSTAGE-eTnpB/ωRNA of interest constructed in example 1 were transferred into E.coli ET12567/pUZ8002 (https:// doi. Org/10.1016/0378-1119 (92) 90549-5), respectively, as follows:

200ng of plasmid pSTAGE-eTnpB and pSTAGE-TnpB, respectively, were added to 100. Mu.L of thawed self-made competent cells E.coli ET12567/pUZ8002, mixed with a light spring tube wall, left on ice for 30 min, then placed immediately on ice for 2-3 min in a 42℃water bath, then an antibiotic-free LB liquid medium was added, incubated at 200rpm,37℃for 1 hour, centrifuged at 5000rpm for 5min, 900. Mu.L of supernatant was discarded, the bacterial body was resuspended in the remaining medium, and applied to LB solid plates containing kanamycin (25. Mu.g/mL), chloramphenicol (12.5. Mu.g/mL) and apramycin (50. Mu.g/mL) and gently smeared with sterile coating bars, after overnight incubation at 37℃a single clone was taken to 20mL LB medium containing kanamycin (25. Mu.g/mL), chloramphenicol (12.5. Mu.g/mL) and apramycin (50. Mu.g/mL) for about 4 min, the liquid medium was centrifuged at 40 rpm for about 2 min, the two centrifugation steps were repeated for 20 min, and the antibiotic-free LB medium was added to the medium.

2.2 Binding transfer and resistance screening

Under non-induction conditions, the plasmid in step 2.1 was transferred into Streptomyces coelicolor A3 (2) by binding, and the specific method is as follows:

Taking Streptomyces spores collected in advance, centrifuging at 5000rpm for 5 minutes, discarding supernatant, resuspending thalli by using 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 the step 2.1 in a 1.5mL centrifuge tube containing 200 mu L of Streptomyces spore suspension, uniformly mixing, taking 200 mu L of mixed liquid, uniformly coating on an MS flat plate, pouring the MS flat plate into a 30 ℃ incubator for culture, covering the surface of the culture medium with apramycin (1 mg) and nalidixic acid (1 mg) after 18 hours, and continuously pouring the MS flat plate into the 30 ℃ incubator until a binder grows (about 5 days) after the surface is dried. 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 the single clone onto ISP2 solid culture medium with plasmid resistance antibiotics and 0.5 mug/mL thiostrepton, inverting, culturing at constant temperature of 30 ℃ until the single clone is visible, picking up a small amount of the single clone to a PCR tube containing 20 mug DMSO, placing the single clone at 100 ℃ for 15 minutes, refrigerating at-20 ℃ for 30 minutes, repeating the steps twice to fully lyse the cells to obtain cell lysate, and then amplifying target site fragments by adopting a Novira 2X RAPID TAQ MASTER Mix (product number: P222-01) kit PCR, wherein the primer design is shown in a table 1, the PCR reaction system is shown in a table 2, and the PCR reaction program is shown in a table 3.

TABLE 1 PCR amplification primers targeting SCO5087 (actI)

Primer(s)	Sequence (5 '-3')	Numbering device
			T-SCO5087-F	GTCGCCTGCTTCGACGCGAT	SEQ ID NO.8
T-SCO5087-R	CCTCCAGCGGAACGTAGTCG	SEQ ID NO.9

TABLE 2PCR reaction System

System of	15μL
		ddH2O	6.0μL
Forward primer (10. Mu.M, SCO 5087-LH-F)	0.6μL
		Reverse primer (10. Mu.M, SCO 5087-R)	0.6μL
2×RapidTaqMasterMix	7.5μL
		Cell lysate (containing DMSO)	0.3μL

Table 3 PCR reaction procedure

The results of the editing of the SCO5087 gene of strain Streptomyces coelicolor A3 (2) were verified by pSTAGE-TnpB and pSTAGE-eTnpB for each of the omega RNAs, and if the target gene SCO5087 (actI) was successfully edited, the theoretical PCR band size at the time of electrophoresis detection was different from that of the wild-type Streptomyces coelicolor A3 (2) strain or the Sanger sequencing result was different from that of the wild-type strain.

The randomly selected monoclonal PCR amplification products were subjected to 1% agarose gel electrophoresis detection, and the detection results of a part of the monoclonal are shown in FIG. 2 and FIG. 3. Lane 1-11 in FIG. 2 and Lane 1-20 in FIG. 3 represent, respectively, partial monoclonals containing pSTAGE-TnpB and pSTAGE-eTnpB of E.coli combined with S.coelicolor A3 (2) and randomly picked after transfer, WT represents the wild-type Streptomyces coelicolor A (2) strain without genetic editing. The results showed that PCR bands of different sizes from the wild type strain were present in the randomly selected resistant strain, and it can be seen that the number of monoclonal antibodies of different sizes from the wild type strain in FIG. 3 were significantly higher than in FIG. 2.

Due to the dependency of the streptomycete on its own NHEJ repair system, random fragment size insertions or deletions can be made after editing. A randomly picked monoclonal PCR product is considered successfully edited if its size differs from the wild type. As with Lane 3 and Lane 4 in FIG. 2, the absence of product after PCR means that the strain was successfully edited, possibly deleting larger fragments. The whole genome re-sequencing of the resistant strain without the PCR product after PCR can prove that the target gene SCO5087 (actI) of the resistant strain without the PCR product after PCR is successfully edited.

All monoclonal PCR products randomly picked after pSTAGE-TnpB and pSTAGE-eTnpB RNA were combined and transferred with s.coelicolor A3 (2) were identified by agarose gel electrophoresis and statistical analysis was performed on the identification results. The results are shown in fig. 4, which shows that the pSTAGE-TnpB x/ωrna editing group has 8.3% of the monoclonal, the target gene SCO5087 is successfully edited, while the pSTAGE-eTnpB x/ωrna editing group has the target gene SCO5087 with the successfully edited monoclonal reaching 41.0% of the ratio, and compared with the pSTAGE-TnpB x/ωrna editing efficiency, the pSTAGE-eTnpB x/ωrna plasmid is proved to be capable of efficiently editing the target gene SCO5087 (actI) in the streptomycete model strain s.carolatora 3 (2).

The PCR products were recovered by purification using a NZU plasmid purification kit (cat# DC 201) and sent to Sanger sequencing analysis by Sanger Biotechnology Inc. of Jin Weizhi, the Sanger sequencing results of resistant strains #4, #13, #29 are shown in FIG. 5 (resistant strains #4 and #13 correspond to Lane 4 and Lane 13, respectively, in FIG. 3), and the sequencing results of FIG. 5 further indicate that the target gene SCO5087 of the resistant strain was successfully edited compared to the wild-type strain.

The above results fully demonstrate that the novel minigene editor pSTAGE-eTnpB RNA can effectively mediate streptomycete genome editing, and the engineered TnpB nuclease and streptomycete minigene editing system can greatly improve the efficiency of gene editing near TTGAT.

The media used in the examples above are as follows:

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 1LddH ₂ O, adjusting 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 suitable for use in streptomyces, wherein the engineered TnpB nuclease has an amino acid site mutation comprising position 41, position 42, position 44, position 45, position 46, position 50, position 51, position 53, position 54, position 56, position 57, position 58, position 64, position 65, position 67, position 69, position 72, position 81, position 84, position 85, position 88, position 91, position 97, position 99 relative to a wild-type TnpB nuclease having the amino acid sequence shown in SEQ ID No. 1.

2. The engineered TnpB nuclease of claim 1, wherein the amino acid site mutation comprises ：E41A、S42A、R44K、Q45E、D46S、M50L、I51T、A53G、A54Q、D56S、K57S、A58E、R64Q、E65A、G67E、A69S、Q81N、R84K、D85N、R88T、Q91K、A97V,K99Q; the amino acid sequence of the engineered TnpB nuclease is set forth in SEQ ID No. 3.

3. The engineered TnpB nuclease of claim 2, wherein the nucleotide sequence of the engineered TnpB nuclease is set forth in SEQ ID No. 4.

4. An engineered TnpB nuclease-mediated streptomycete minigene editing system comprising the engineered TnpB nuclease of any one of claims 1-3, and a guide RNA.

5. The streptomycete minigene editing system of claim 4, wherein the guide RNA comprises an RNA backbone, a gene targeting segment, and a hepatitis delta virus ribozyme.

6. The streptomycete minigene editing system according to claim 5, wherein the nucleotide sequence of the RNA backbone is shown as SEQ ID NO.5, the gene targeting segment is positioned at the 3' end of the RNA backbone and is a nucleic acid fragment with a length of 12-40bp after a TAM sequence on a target gene, and the nucleotide sequence of the hepatitis delta virus ribozyme is shown as SEQ ID NO. 7.

7. A recombinant expression plasmid vector for expressing the Streptomyces minigene editing system according to claim 4.

8. A method of constructing a Streptomyces minigene editing system according to claim 4, comprising constructing a gene editing plasmid containing an apramycin resistance selection marker, said gene editing plasmid being constructed by inserting said engineered TnpB nuclease and said guide RNA on the basis of an E.coli-Streptomyces shuttle plasmid.

9. Use of the streptomycete minigene editing system of claim 4 for mediating streptomycete gene editing.

10. The use according to claim 9, wherein the method of mediating the editing of a streptomyces gene comprises the steps of:

S1, under the non-induction condition, transforming a gene editing plasmid into a target host, and obtaining a transformant carrying the gene knockout/plasmid entry through a DNA repair mechanism of streptomycete in vivo and antibiotic screening of plasmid related resistance;

S2, streaking and inoculating the transformant in the step S1 to a culture medium plate containing plasmid resistance antibiotics and promoter inducers, and culturing at constant temperature until the single clone is visible;

S3, randomly selecting the monoclonal in the step S2, and performing colony PCR verification to obtain the edited strain.