Recombination system and its uses

Abstract

The present invention belongs to the field of gene editing, and specifically relates to a recombination system and its use. Specifically, the present invention provides a recombineering system suitable for mycobacteria, which comprises an expression system of a gene sequence encoding a RecE protein or its homolog and / or a RecT protein or its homolog. The RecE / RecT system disclosed in the present invention shows a very high (>90%) gene replacement rate in M.neoaurum strains. Another aspect of the present invention relates to a scarless genome editing tool suitable for mycobacteria based on a newly designed RecE / RecT system and a DNA cutting system used in combination. Scarless and efficient gene knockout and knock-in are achieved in M.neoaurum strains.

Description

Technical Field

[0001] The present invention belongs to the field of gene editing, and in particular relates to a recombination system and uses thereof. Background Art

[0002] Genetic modification is a fundamental method for studying gene function and constructing enzymes or strains with new properties. The development of genetic tools, especially those that can efficiently generate desired modifications in a targeted manner, has always been a hot area in biological research.

[0003] Over the past two decades, a technology called recombineering has been developed that can modify DNA sequences in a precise, rapid, and efficient manner without cutting or destroying the target DNA molecule (Thomason et al. 2023). This is a homologous recombination process mediated by recombinant proteins from bacteriophages. The process utilizes a segment of DNA (donor) that contains the desired mutation modification flanked by regions (homologous arms) that are homologous to the target DNA molecule. The recombinant proteins derived from bacteriophages catalyze the recombination reaction so that the sequence between the regions of the target molecule that bind to the two homologous arms of the donor is replaced by the donor sequence. Homologous arms as short as 30-50 nucleotides can be used for recombineering. The donor is a DNA fragment that can be double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). The target molecule can be a bacterial chromosome, a bacterial artificial chromosome, a plasmid, and a bacteriophage.

[0004] There are two key components in the recombineering system derived from bacteriophage: 5'-3' exonuclease and ssDNA annealing protein. There are two phage recombineering systems commonly used in Escherichia coli, namely the λ Red system, which consists of three proteins Exo (5'-3' exonuclease), Beta (ssDNA annealing protein) and Gam (RecBCD exonuclease inhibitor protein); and the Rac Prophage system, which consists of two proteins RecE (5'-3' exonuclease) and RecT (ssDNA annealing protein). The 5'-3' exonuclease can degrade a single strand of the linear dsDNA donor, exposing the 3'ssDNA tail end. The ssDNA annealing protein binds to the exposed ssDNA tail end, promoting the binding of the ssDNA tail end to the target molecule region homologous to its sequence. Theoretically, when a dsDNA donor is used, both the 5'-3' exonuclease and the ssDNA annealing protein participate in the recombination reaction; when an ssDNA donor is used, the ssDNA annealing protein can mediate the recombination without the need for the 5'-3' exonuclease. Existing literature has shown that the expression of phage-derived recombinant proteins in Escherichia coli can increase the recombination efficiency by 10 to 100 times (Zhang et al., 1998; Yu et al., 2000).

[0005] Genetic modifications, such as gene knockout, replacement, deletion, insertion, and point mutation, can all be achieved through recombineering. In most cases, dsDNA is used as a donor. ssDNA donors can also achieve gene mutations, but due to technical limitations, this strategy can only achieve very short sequence changes because these changes need to be contained in a synthetic single-stranded nucleotide (ssDNA donor) sequence; and when the target gene has no screenable phenotype, there is no good way to screen the mutant product.

[0006] In addition to being used alone, the recombineering system can also be used in conjunction with other gene modification systems to improve the overall gene editing efficiency by increasing the efficiency of homologous recombination. One example is the coupling of the recombineering system with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Associated Protein (Cas) system. In Escherichia coli, the λRed recombineering system coupled with the CRISPR-Cas9 system can achieve efficient DNA deletions of up to 19 kb and DNA insertions of up to 3 kb (Pyne et al., 2015).

[0007] Despite their important role in genetic modification, phage-derived recombineering systems are not broadly available to microbial hosts. They work in a few interrelated bacterial species but perform poorly in less related bacterial species. Fortunately, scientists have discovered functional RecE / T systems in some bacteria other than E. coli, such as Corynebacterium (Chang et al., 2021), Bacillus sutbilis (Sun et al., 2015), and Burkholderia (Li et al., 2021). RecE / T-assisted CRISPR-Cas genome editing tools have also been developed for some bacteria, such as C. glutamicum (Wang et al., 2018) and Lactobacillus (Huang et al., 2019).

[0008] The mycobacterium genus is a diverse group of organisms that includes more than 180 species, including pathogenic species such as Mycobacterium tuberculosis and Mycobacterium leprae, as well as industrially important species such as Mycolicibacterium smegmatis and Mycolicibacterium neoaurum. Developing effective genetic manipulation tools for mycobacteria is of great importance.

[0009] In 2007, van Kessel et al. developed a recombineering tool for mycobacteria. The RecE / RecT homologues (gp60 / gp61 proteins) from the mycobacteriophage Che9c, which infects mycobacteria, were found to mediate DNA recombination in strains of M. smegmatis and M. tuberculosis. In the presence of plasmid pJV53 encoding the Che9c gp60 / gp61 proteins, targeted gene replacement rates in M. smegmatis and M. tuberculosis strains were >90% (van Kessel et al., 2007). In addition, Che9cgp61 and a RecT homolog (gp43) from another mycobacteriophage, Halo, were shown to be efficient in ssDNA-mediated recombination in M. smegmatis and M. tuberculosis (van Kessel et al. 2008a, 2008b), but since then, further validation or development of mycobacterial recombineering systems has been lacking.

[0010] Although pJV53-mediated recombination is reported to be highly efficient in the literature, the inventors found in gene replacement experiments on M. neoaurum strains that the recombination products that did not meet the expected results dominated the recombination products, resulting in a very low gene replacement rate (<20%).

[0011] In addition, although the traditional gene replacement method containing the RecE / RecT system can achieve precise gene knockout and knock-in, it leaves antibiotic markers in the genome. Due to the limited available antibiotic markers, this will limit further genome modifications. And in the fermentation industry, the presence of resistant microorganisms and genes encoding resistance has raised concerns about environmental and public health issues.

[0012] Therefore, there is an urgent need to develop a new recombination system that can achieve high gene replacement rates and scarless genome editing. Summary of the invention

[0013] In order to solve the problems existing in the prior art, the present disclosure aims to provide a recombination system and its use.

[0014] In order to solve the above technical problems, the present disclosure adopts the following technical solutions:

[0015] In one aspect, the present disclosure provides a recombinant engineering system suitable for mycobacteria, which comprises an expression system of a gene sequence encoding a RecE protein or a homologue thereof and / or a RecT protein or a homologue thereof.

[0016] In some embodiments, the expression system comprises the gene sequence of optimized RecE protein and RecT protein. In some embodiments, the expression system comprises the gene sequence of optimized RecE protein or RecT protein. In some embodiments, the expression system comprises the gene sequence of optimized RecE protein homologue and RecT protein homologue. In some embodiments, the expression system comprises the gene sequence of optimized RecE protein homologue or RecT protein homologue.

[0017] In some embodiments, the RecE protein or a homolog thereof and the RecT protein or a homolog thereof are derived from mycobacteriophages, prophages or bacteria.

[0018] In some embodiments, the RecE protein or a homolog thereof and the RecT protein or a homolog thereof are from the same phage, prophage or bacterium.

[0019] In some embodiments, the RecE protein or a homolog thereof and the RecT protein or a homolog thereof are from different bacteriophages, prophages or bacteria.

[0020] In some embodiments, the expression system comprises only the gene sequence of the RecE protein or its homologue. In some embodiments, the RecE protein or its homologue is selected from the amino acid sequence shown in any one of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24 or any variant thereof.

[0021] In some embodiments, the expression system RecE protein or its homolog is selected from an amino acid sequence shown in any one of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, or a variant having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity with any of the aforementioned amino acid sequences.

[0022] In some embodiments, the expression system RecE protein or its homologue is selected from the amino acid sequence shown in any one of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, or a variant having at least 80%, 85%, 90%, 95% or 99% sequence identity with any of the aforementioned amino acid sequences. In some preferred embodiments, the expression system RecE protein is selected from the amino acid sequence shown in any one of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, or a variant having at least 80% sequence identity with any of the aforementioned amino acid sequences. In some preferred embodiments, the RecE protein or its homologue is selected from the amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 18, 20, 22, 24.

[0023] In some embodiments, the expression system comprises only the gene sequence of the RecT protein or its homologue. In some embodiments, the RecT protein or its homologue is selected from the amino acid sequence shown in any one of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25 or any variant thereof.

[0024] In some embodiments, the RecT protein or its homolog is selected from an amino acid sequence as shown in any one of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, or a variant having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity with any of the aforementioned amino acid sequences.

[0025] In some embodiments, the RecT protein or its homologue is selected from the amino acid sequence as shown in any one of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, or a variant having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity with any of the aforementioned amino acid sequences. In some preferred embodiments, the RecT protein is selected from the amino acid sequence as shown in any one of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, or a variant having at least 64% sequence identity with any of the aforementioned amino acid sequences. In some preferred embodiments, the RecT protein or its homologue is selected from the amino acid sequence having 64% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 19, 21, 23, 25.

[0026] In some embodiments, the RecT protein has an amino acid sequence as shown in SEQ ID NO: 19. In some embodiments, the RecT protein is encoded by a nucleotide sequence as shown in SEQ ID NO:33.

[0027] In some embodiments, the expression system comprises a combination of RecE protein or its homologue and RecT protein or its homologue. In some embodiments, the RecE protein or its homologue and RecT protein or its homologue are selected from any one of the following combinations:

[0028] (1) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 12 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 13 or any variant thereof;

[0029] (2) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 14 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 15 or any variant thereof;

[0030] (3) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 16 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 17 or any variant thereof;

[0031] (4) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 18 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 19 or any variant thereof;

[0032] (5) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 20 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 21 or any variant thereof;

[0033] (6) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 22 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 23 or any variant thereof;

[0034] (7) A RecE protein having an amino acid sequence as shown in SEQ ID NO: 24 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 25 or any variant thereof.

[0035] In some preferred embodiments, the RecE protein or its homologue and the RecT protein or its homologue are selected from any one of the following combinations:

[0036] (1) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 12, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 13;

[0037] (2) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 14, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 15;

[0038] (3) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 16, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 17;

[0039] (4) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 18, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 19;

[0040] (5) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 20 or a variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 21 or a variant thereof;

[0041] (6) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 22 or a variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 23 or a variant thereof;

[0042] (7) A RecE protein having an amino acid sequence as shown in SEQ ID NO: 24 or a variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 25 or a variant thereof.

[0043] In some preferred embodiments, the RecE protein or its homologue and / or the RecT protein or its homologue is selected from any one of the following combinations:

[0044] (1) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 18 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 19 or any variant thereof;

[0045] (2) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 20 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 21 or any variant thereof;

[0046] (3) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 22 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 23 or any variant thereof;

[0047] (4) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 24 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 25 or any variant thereof;

[0048] (5) A RecT protein having an amino acid sequence as shown in SEQ ID NO: 19 or any variant thereof.

[0049] In some preferred embodiments, the RecE protein or its homologue and / or the RecT protein or its homologue is selected from any one of the following combinations:

[0050] (1) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 18, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 19;

[0051] (2) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 20, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 21;

[0052] (3) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 22, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 23;

[0053] (4) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 24, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 25;

[0054] (5) A RecT protein having the amino acid sequence shown in SEQ ID NO: 19.

[0055] In some preferred embodiments, the RecE protein or its homologue and the RecT protein or its homologue are selected from any one of the following combinations:

[0056] (1) a RecE protein encoded by the nucleotide sequence shown in SEQ ID NO: 32, and a RecT protein encoded by the nucleotide sequence shown in SEQ ID NO: 33;

[0057] (2) a RecE protein encoded by the nucleotide sequence shown in SEQ ID NO: 34, and a RecT protein encoded by the nucleotide sequence shown in SEQ ID NO: 35;

[0058] (3) a RecE protein encoded by the nucleotide sequence shown in SEQ ID NO: 36, and a RecT protein encoded by the nucleotide sequence shown in SEQ ID NO: 37;

[0059] (4) The RecE protein encoded by the nucleotide sequence shown in SEQ ID NO:38, and the RecT protein encoded by the nucleotide sequence shown in SEQ ID NO:39.

[0060] In some embodiments, the expression system is integrated directly into the host genome.

[0061] In some embodiments, the expression system is encoded by a mobile genetic element. In some embodiments, the mobile genetic element is a plasmid, a transposon or a bacteriophage. In some preferred embodiments, the mobile genetic element is a plasmid. In some embodiments, the plasmid further comprises a promoter.

[0062] In some embodiments, the promoter is an inducible promoter or a constitutive promoter.

[0063] In some embodiments, the inducible promoter is a tetracycline-inducible promoter, a nitrile-inducible promoter, a thiostrepton-inducible promoter, or an acetamide-inducible promoter.

[0064] In some embodiments, the constitutive promoter is an hsp60 promoter, a pms promoter, a trp promoter, a Psmyc promoter, or a Pimyc promoter.

[0065] In some embodiments, recE gene and recT gene are transcribed from promoter separately respectively.In some embodiments, these two genes are organized in a cis operon, and its transcription is driven by the same promoter.The order of gene in the operon may affect the gene expression level.In some embodiments, recE gene is at recT gene upstream, and in some embodiments, recT gene is at recE gene upstream.

[0066] In some embodiments, the plasmid comprises a nucleotide sequence selected from any one of SEQ ID NOs: 40-59.

[0067] In some embodiments, the plasmid comprises a nucleotide sequence selected from any one of SEQ ID NOs: 49-59.

[0068] In some embodiments, the recombineering system further comprises a first recombination donor, which comprises an upstream homology arm and a downstream homology arm from the 5' end to the 3' end, respectively; optionally, a mutation sequence is further comprised between the upstream homology arm and the downstream homology arm, and the mutation sequence is used to replace the gene editing target sequence of the mycobacterium.

[0069] In some embodiments, the first recombination donor is double-stranded DNA or single-stranded DNA.

[0070] In some embodiments, the first recombinant donor further comprises a sequence homologous to the upstream homology arm, located between the upstream homology arm and the downstream homology arm. In some embodiments, the first recombinant donor further comprises a cleavage site for at least one DNA cleavage system, located between the upstream homology arm and the sequence homologous to the upstream homology arm.

[0071] In some embodiments, the first recombinant donor also comprises a sequence homologous to the downstream homology arm, between the upstream homology arm and the downstream homology arm. In some embodiments, the first recombinant donor also comprises a cleavage site of at least one DNA cutting system, between the downstream homology arm and the sequence homologous to the downstream homology arm.

[0072] In some embodiments, the first recombinant donor further comprises a sequence homologous to a fragment located upstream of the upstream homology arm binding region in the target molecule, which is located between the upstream homology arm and the downstream homology arm. In some embodiments, the first recombinant donor further comprises a cleavage site for at least one DNA cleavage system, which is located between the upstream homology arm and the sequence homologous to a fragment located upstream of the upstream homology arm binding region in the target molecule.

[0073] In some embodiments, the first recombinant donor further comprises a sequence homologous to a fragment located downstream of the downstream homology arm binding region in the target molecule, which is located between the upstream homology arm and the downstream homology arm. In some embodiments, the first recombinant donor further comprises a cleavage site for at least one DNA cleavage system, which is located between the downstream homology arm and the sequence homologous to a fragment located downstream of the downstream homology arm binding region in the target molecule.

[0074] In another aspect, the present disclosure provides a tool kit for scarless genome editing, comprising the aforementioned recombineering system, and optionally a DNA cleavage system.

[0075] In some embodiments, the DNA cleavage system is selected from a homing endonuclease, a CRISPR-Cas system, a transcription activator-like effector nuclease (TALENs) or a zinc finger nuclease (ZFNs). In some embodiments, the homing endonuclease is I-SceI.

[0076] In some embodiments, the tool box further comprises a second recombination donor for guiding DNA repair. In some embodiments, the second recombination donor comprises an upstream homology arm and a downstream homology arm from the 5' end to the 3' end, respectively. In some embodiments, the second recombination donor further comprises a mutant sequence located in the middle of the upstream homology arm and the downstream homology arm.

[0077] In another aspect, the present disclosure provides a genetically engineered bacterium comprising the aforementioned recombinant engineering system or the aforementioned tool kit.

[0078] In another aspect, the present disclosure provides a method for performing scarless genome editing using the aforementioned recombineering system or the aforementioned tool kit, comprising the following steps:

[0079] Providing a host cell, wherein the host cell comprises the aforementioned recombineering system or the aforementioned tool kit;

[0080] transforming the first recombinant donor into a host cell;

[0081] Using a recombineering system comprising RecE protein or its homolog and / or RecT protein or its homolog to mediate the recombination of the first recombination donor and the gene editing target molecule;

[0082] Optionally, the method further comprises the steps of:

[0083] Targeted DNA cleavage using a DNA cleavage system.

[0084] The first recombinant donor or the second recombinant donor is used to guide DNA repair.

[0085] In a preferred embodiment, the scarless gene editing method is achieved by the combined action of a recombineering system comprising RecE protein and / or RecT protein and a DNA cutting system. In a preferred embodiment, the scarless gene editing is carried out in three steps, the first step is the first recombinant donor (including upstream homology arms, downstream homology arms and mutant sequences therebetween), mediated by RecE / RecT, and recombined with the target molecule. The second step is the cutting of the target molecule cutting site catalyzed by the DNA cutting system. The third step is DNA repair guided by the first donor or the second donor.

[0086] In some embodiments, in the first step, the first recombinant donor includes at least one cleavage site between the upstream homology arm and the downstream homology arm, which is recognized by the DNA cutting system. In some embodiments, the first recombinant donor includes more than one cleavage site, located between the upstream homology arm and the downstream homology arm. In some embodiments, the first recombinant donor includes a mutant sequence intended for knock-in, located between the upstream homology arm and the cleavage site, or / and between the downstream homology arm and the cleavage site. In some embodiments, the first recombinant donor also includes a sequence homologous to the upstream homology arm, located between the upstream homology arm and the downstream homology arm, and there is at least one cleavage site between the upstream homology arm and the sequence homologous to the upstream homology arm. In some embodiments, the first recombinant donor also includes a sequence homologous to the downstream homology arm, located between the upstream homology arm and the downstream homology arm, and there is at least one cleavage site between the downstream homology arm and the sequence homologous to the downstream homology arm. In some embodiments, the first recombinant donor also includes a sequence homologous to a fragment located upstream of the upstream homology arm binding region in the target molecule, located between the upstream homology arm and the downstream homology arm, and there is at least one cleavage site between the upstream homology arm and the sequence homologous to a fragment located upstream of the upstream homology arm binding region in the target molecule. In some embodiments, the first recombinant donor also includes a sequence homologous to a fragment located downstream of the downstream homology arm binding region in the target molecule, located between the upstream homology arm and the downstream homology arm, and there is at least one cleavage site between the downstream homology arm and the sequence homologous to a fragment located downstream of the downstream homology arm binding region in the target molecule. In a specific embodiment, the first recombinant donor includes an upstream homology arm, a sequence intended for knock-in, a sequence homologous to the downstream homology arm, a cleavage site, an antibiotic marker, a cleavage site, and a downstream homology arm in sequence from the 5' to the 3' end. In a specific embodiment, the first recombinant donor includes, from 5' to 3' end, an upstream homology arm, a sequence intended for knock-in, a sequence homologous to a fragment located downstream of the downstream homology arm binding region in the target molecule, a cleavage site, an antibiotic marker, a cleavage site, and a downstream homology arm. In a specific embodiment, the first recombinant donor includes, from 5' to 3' end, an upstream homology arm, a cleavage site, an antibiotic marker, a cleavage site, a sequence homologous to the upstream homology arm, a sequence intended for knock-in, and a downstream homology arm. In a specific embodiment, the first recombinant donor includes, from 5' to 3' end, an upstream homology arm, a cleavage site, an antibiotic marker, a cleavage site, a sequence homologous to a fragment located upstream of the upstream homology arm binding region in the target molecule, a sequence intended for knock-in, and a downstream homology arm.

[0087] In some embodiments, in the second step, the DNA cutting system is encoded by a mobile gene element, such as a plasmid. In certain embodiments, the DNA cutting system is located on the same mobile gene element as the RecE / RecT system. In certain embodiments, the DNA cutting system is integrated into the host genome. The DNA cutting system can be constitutively expressed or induced to express. In certain embodiments, the DNA cutting system is selected from a homing endonuclease, a CRISPR-Cas system, a transcription activator-like effector nuclease (TALENs) or a zinc finger nuclease (ZFNs). In certain embodiments, the DNA cutting system is a CRISPR-Cas system. In certain embodiments, the DNA cutting system is a homing endonuclease.

[0088] In some embodiments, in the third step, a second donor molecule including an upstream homology arm and a downstream homology arm is provided to guide DNA repair. In some embodiments, the second donor also includes a mutant sequence intended for knock-in between the upstream homology arm and the downstream homology arm. In some embodiments, a second donor is not provided, and DNA repair occurs by the recombination between the sequence homologous to the upstream homology arm and the upstream homology arm of the first donor that has been integrated into the target molecule. In some embodiments, a second donor is not provided, and DNA repair occurs by the recombination between the sequence homologous to the downstream homology arm and the downstream homology arm of the first donor that has been integrated into the target molecule. In some embodiments, a second donor is not provided, and DNA repair occurs by the recombination between the sequence homologous to the downstream homology arm and the downstream homology arm of the first donor that has been integrated into the target molecule. In some embodiments, a second donor is not provided, and DNA repair occurs by the recombination between the sequence homologous to a fragment located upstream / downstream of the upstream / downstream homology arm binding region in the target molecule encoded by the first donor in the above-mentioned first step and the homologous sequence in the target molecule.

[0089] In some embodiments, the method comprises the following steps:

[0090] Providing a host cell, wherein the host cell comprises the aforementioned recombineering system or the aforementioned tool kit;

[0091] Transforming a first recombinant donor into a host cell, wherein the first recombinant donor comprises, from the 5' end to the 3' end, an upstream homology arm, optionally a mutation sequence, a downstream homology arm, and at least one cleavage site located between the upstream homology arm and the downstream homology arm, respectively, and the cleavage site is recognized by a DNA cleavage system;

[0092] Using a recombineering system comprising RecE protein and / or RecT protein to mediate the recombination of the first recombinant donor and the gene editing target molecule;

[0093] Utilize DNA cleavage system for targeted DNA cleavage;

[0094] The second recombinant donor is used to guide DNA repair, wherein the second recombinant donor comprises an upstream homology arm and a downstream homology arm from the 5' end to the 3' end, respectively, and optionally, a mutation sequence located between the upstream homology arm and the downstream homology arm.

[0095] In some embodiments, the method comprises the following steps:

[0096] Providing a host cell, wherein the host cell comprises the aforementioned recombineering system or the aforementioned tool kit;

[0097] Transforming a first recombinant donor into a host cell, wherein the first recombinant donor comprises, from the 5' end to the 3' end, an upstream homology arm, optionally a mutant sequence, a downstream homology arm, and a sequence homologous to the upstream or downstream homology arm located between the upstream homology arm and the downstream homology arm, and at least one cleavage site located between the upstream or downstream homology arm and the sequence homologous to the upstream or downstream homology arm, and the cleavage site is recognized by a DNA cleavage system;

[0098] Using a recombineering system comprising RecE protein and / or RecT protein to mediate the recombination of the first recombinant donor and the gene editing target molecule;

[0099] Utilize DNA cleavage system for targeted DNA cleavage;

[0100] DNA repair is directed by recombination between homologous sequences in the first recombination donor and the upstream or downstream homology arms.

[0101] In some embodiments, the method comprises the following steps:

[0102] Providing a host cell, wherein the host cell comprises the aforementioned recombineering system or the aforementioned tool kit;

[0103] Transforming a first recombinant donor into a host cell, wherein the first recombinant donor comprises, from the 5' end to the 3' end, an upstream homology arm, an optionally mutated sequence, a downstream homology arm, and a sequence between the upstream homology arm and the downstream homology arm homologous to a fragment of the target molecule located upstream of the upstream homology arm binding region, and at least one cleavage site between the upstream homology arm and the sequence homologous to a fragment of the target molecule located upstream of the upstream homology arm binding region, or a sequence between the upstream homology arm and the downstream homology arm homologous to a fragment of the target molecule located downstream of the downstream homology arm binding region, and at least one cleavage site between the downstream homology arm and the sequence homologous to a fragment of the target molecule located downstream of the downstream homology arm binding region, and the cleavage site is recognized by a DNA cleavage system;

[0104] Using a recombineering system comprising RecE protein and / or RecT protein to mediate the recombination of the first recombinant donor and the gene editing target molecule;

[0105] Utilize DNA cleavage system for targeted DNA cleavage;

[0106] DNA repair is guided by recombination between homologous sequences in the first recombinant donor and corresponding homologous sequences upstream or downstream in the target molecule.

[0107] In some embodiments, the DNA cleavage system is selected from a homing endonuclease, a CRISPR-Cas system, a transcription activator-like effector nuclease (TALENs), or a zinc finger nuclease (ZFNs).

[0108] In some embodiments, the homing endonuclease is I-Scel.

[0109] In some embodiments, the host cell is a mycobacterium microorganism. In some embodiments, the mycobacterium is a fast-growing mycobacterium, including Mycobacterium smegmatis, Mycobacterium fortuitum, Mycobacterium flavosum, and Mycobacterium neogoldenum. In some embodiments, the mycobacterium is Mycobacterium neogoldenum. In some embodiments, the mycobacterium is a slow-growing mycobacterium, including Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium complex, Mycobacterium surga, Mycobacterium malmo, Mycobacterium scrofulae, and Mycobacterium scrofulum. In some embodiments, the mycobacterium is M.neoaurum strain NRRL B-3805.

[0110] The present disclosure has at least the following beneficial effects:

[0111] The new RecE / RecT recombination system disclosed in the present invention achieves a very high gene replacement rate (>90%) in gene replacement experiments mediated by dsDNA donors, and also achieves efficient replacement effects in point mutation experiments of genes in the genome mediated by ssDNA donors. In addition, by combining the representative new recombination system with the DNA cutting system, an efficient scarless genome editing tool has been developed. BRIEF DESCRIPTION OF THE DRAWINGS

[0112] Figure 1 A schematic diagram of scarless gene editing using the RecE / RecT system is shown. Figure 1 A is the first step, in which the nucleotide donor molecule composed of the upstream homology arm, the downstream homology arm and the intermediate antibiotic marker is recombined with the target molecule, mediated by the RecE / RecT system. Figure 1 B is the second step of gene editing, in which the DNA cleavage at the cleavage site is catalyzed by the DNA cleavage system. Figure 1 C is the third step of gene editing, DNA repair assisted by the RecE / RecT system.

[0113] Figure 2 A schematic diagram of the MyAD_23825 gene replacement in the B-3805 strain is shown. Figure 2 A is a schematic diagram of the parent B-3805 genome, with the upstream homology arm binding region and the downstream homology arm binding region on both sides of the gene MyAD_23825. Figure 2 B is a schematic diagram of the genome of the expected product after gene replacement, where the MyAD_23825 gene sequence is replaced with the gentamicin resistance cassette in the first donor molecule. The position of the primers used for colony PCR detection is shown in the figure.

[0114] Figure 3 The agarose gel electrophoresis diagram of the MyAD_23825 gene replacement PCR detection results mediated by pJV53 of B-3805 strain is shown. 24 transformant colonies ( Figure 3 A) and 24 transformant colonies obtained by transformation of competent cells prepared under acetamide induction conditions ( Figure 3 B) As PCR template, use Figure 2 The upstream primer pair (P1+P2) and the downstream primer pair (P3+P4) shown are respectively used for PCR. Each colony is represented by numbers 1-24. Each colony has 2 PCR products, which are electrophoresed side by side in agarose gel. For each colony, the product using the upstream primer pair (P1+P2) is on the left (one of the positions representing the product is marked as "a"), and the product using the downstream primer pair (P3+P4) is on the right (one of the positions representing the product is marked as "b"). As can be seen from the electrophoretogram, the transformant colonies 4, 6, 9 under the uninduced condition and the transformant colonies 2 and 8 under the induced condition all show the expected PCR products when using the two primer pairs.

[0115] Figure 4 Strain S1 ( Figure 4 A) and S2( Figure 4 B) Schematic diagram of the region near the genomic modification site. The GFP reporter strain S2 was constructed by integrating the GFP expression cassette into the genome of the S1 strain.

[0116] Figure 5 The suicide plasmid map used to generate the S2 strain is shown.

[0117] Figure 6The transformation result of the MyAD_06390 gene recombination of the use ssDNA donor of RecET mediation is shown.Donor XXIII is transformed into S3 bacterial strain, and transformant is laid on the solid medium containing 50 mg / L kanamycin sulfate and 100 mg / L streptomycin sulfate (right culture dish).As negative control, the S3 competent cells that are not transformed with donor XXIII of the same bacterial amount are laid on the solid medium containing 50 mg / L kanamycin sulfate and 100 mg / L streptomycin sulfate (left culture dish).

[0118] Figure 7 The sequence alignment results of the RecET-mediated ssDNA recombination products are shown. 12 donor XXIII transformants were randomly selected from the selective solid medium for PCR detection. The sequencing results of the obtained PCR products were compared with the parental sequences to confirm that all transformants showed the same mutation introduced by the donor XXIII recombination.

[0119] Figure 8 The map of pBotaET37 is shown.

[0120] Fig. 9 The map of pBotaET38 is shown.

[0121] Fig.10 Eight transformants each of pBotaET37 (left column) and pBotaET38 (right column) grown in YPD medium are shown. None of the pBotaET37 transformants were fluorescent, while seven of the pBotaET38 transformants were red fluorescent.

[0122] Fig.11 A schematic diagram of the target region in the S7 genome (A) and a schematic diagram of the expected editing products of genome editing mediated by pBotaET37 (B) and pBotaET38 (C) are shown.

[0123] Fig.12 The electrophoresis results are shown. Fig.12 A is the electrophoresis result of the products of PCR detection of 8 pBotaET37 transformants using primers P11 and P12; Fig.12 B is the electrophoresis result of the products of PCR detection of 8 pBotaET38 transformants using P11 and P12 primers (marked as AH) and P11 and P13 primers (marked as A'-H'). The colonies whose PCR product size is consistent with the expected product are circled.

[0124] Fig.13 The sequence alignment results are shown. Fig.13 A is the alignment of the sequencing results of the PCR detection products (P11+P12) of two random pBotaET37 transformants with the expected editing product sequences. Fig.13 B is the alignment of the sequencing results of the PCR detection products (P11+P12) of two random pBotaET38 transformants with the expected editing product sequences.

[0125] Fig.14 The map of pBotaET39 is shown.

[0126] Fig.15 A schematic diagram of the target region in the S8 genome (A) and a schematic diagram of the expected product of pBotaET39-mediated genome editing (B) are shown. U represents the binding region of the upstream homology arm of Donor XXVII; H represents the sequence homologous to the downstream homology arm of Donor XXVII; D represents the binding region of the downstream homology arm of Donor XXVII.

[0127] Fig.16 The electrophoresis results of the products of PCR detection of eight pBotaET39 transformants using P14 and P15 primers are shown.

[0128] Fig.17 The sequencing results of five pBotaET39 transformant PCR detection products are aligned with the expected editing product sequence.

[0129] Fig.18 A schematic diagram of the target region in the S11 genome (A) and a schematic diagram of the expected product of genome editing mediated by pBotaET39 (B) are shown. U represents the binding region of the upstream homology arm of Donor XXVIII; H represents a sequence homologous to the binding region of the downstream homology arm of Donor XXVIII; D represents the binding region of the downstream homology arm of Donor XXVIII.

[0130] Fig.19 Shown are the electrophoresis results of PCR detection products of six Donor XXVIII transformants. Fig.19 A shows the PCR products using primers P16 and P17; Fig.19 B shows the PCR products using primers P19 and P17; Fig.19 C shows the PCR product using P16 and P18 primers.

[0131] Fig. 20 Shown are the electrophoresis results of PCR detection products of six pBotaET39 transformants.

[0132] Fig.21 The sequencing results of the PCR detection products of two pBotaET39 transformants are aligned with the expected editing product sequences. DETAILED DESCRIPTION

[0133] I. Definitions

[0134] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, chemistry-related terms and laboratory procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0135] The terms "including" and "having" and any variations thereof in the present disclosure are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product or device comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps that are not listed, or may optionally include other steps that are inherent to these processes, methods, products or devices. The "plurality" mentioned in the present disclosure refers to two or more. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the previously associated objects are in an "and / or" relationship.

[0136] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the terms listed before the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also includes BA, CA, CB, ACB, CBA, BCA, BAC, or CAB if the order is important in the particular context. Continuing with this example, combinations containing repetitions of one or more terms or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. The skilled artisan will understand that, unless otherwise apparent from the context, there is generally no limit to the number of terms or terms in any combination.

[0137] In the description of this document, reference is made to “some embodiments”, “some implementation schemes” or “some implementation plans”, which describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0138] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and where it does not occur.

[0139] The term "recombinant expression vector" is a recombinant or synthetically produced nucleic acid construct having a series of specific nucleic acid factors that allow transcription of a specific polynucleotide sequence in a host cell. The expression vector can be a plasmid, a viral genome, or a portion of a nucleic acid fragment. Typically, the expression vector includes a polynucleotide to be transcribed operably linked to a promoter.

[0140] The term "mobile genetic elements" (MGEs) refers to a class of DNA sequences that can move in the genome, or genetic elements that can be transferred from one host or replica to another. They are widely present in different species and play an important role in evolution and genome diversity. In addition to plasmids, mobile elements also include transposons, certain bacterial viruses (such as lambda phage), integrative and conjugative elements (ICEs), genomic islands, etc.

[0141] The term "promoter" refers to a special sequence on DNA, located in the upstream region of a gene, which is responsible for initiating and regulating the transcription process of the gene. The promoter sequence binds to RNA polymerase or other transcription factors to form a transcription initiation complex, thereby determining the starting point and transcription efficiency of gene transcription. Promoters can be divided into constitutive promoters and inducible promoters. Promoters that are always active are called constitutive promoters. Inducible promoters have two states: on and off. Inducible promoters can be regulated by positive induction and negative induction mechanisms. When the activator is not bound to the promoter, the positive inducible promoter is in the "off" state; after the inducer binds to the activator, the activator can bind to the positive inducible promoter, turn it on and start transcription. When negative inducible promoters bind to repressors, they are in the "off" state; when the inducer binds to the repressor, the repressor will leave the promoter and start transcription.

[0142] Inducible promoters include, but are not limited to, promoters described by Brautaset et al. (2009). Specifically, inducible promoters validated for use in mycobacteria include: nitrile-inducible promoters and thiostrepton-inducible promoters described by Sassetti CM (2009), acetamide-inducible promoters used by van Kessel et al. (2007), and anhydrotetracycline-inducible promoters described by Ehrt et al. (2005), as well as derivatives of the above promoters. In some embodiments, an inducible promoter is used to drive the expression of RecE / RecT proteins.

[0143] Constitutive promoters include, but are not limited to, natural promoters derived from bacterial genomes; synthetic promoters described by Davis et al. (2011); and synthetic promoters listed on the igem website (https: / / parts.igem.org / Promoters / Catalog / Constitutive). Specifically, constitutive promoters validated for use in mycobacteria include, but are not limited to, promoters described by Sun et al. (2020), such as hsp60 promoter, pms promoter, trp promoter, etc.; promoters described by Chang et al. (2009), such as Psmyc and Pimyc. In some embodiments, a constitutive promoter is used to drive the expression of RecE / RecT protein.

[0144] The ribosome binding site (RBS), also known as the Shine-Dalgarno sequence, is a purine-rich sequence in the 5' untranslated region (UTR) of the transcript that is complementary to the UCCU core sequence at the 3'-end of the 16S rRNA. The RBS promotes efficient and accurate translation of mRNA. In some embodiments, the RBS sequence of the recE / recT gene is optimized to promote or coordinate RecE / RecT expression. The RBS sequence can be selected from, but is not limited to: a natural RBS sequence derived from a bacterial genome; a synthetic RBS sequence described by Sun et al. (2020); and a synthetic RBS sequence listed on the igem website https: / / parts.igem.org / Ribosome_Binding_Sites / Prokaryotic / Constitutive / Anderson.

[0145] RecE and RecT are two proteins associated with the homologous recombination process and play an important role in bacterial DNA repair and genetic engineering. RecE is a 5'-3' exonuclease and RecT is a single-stranded DNA annealing protein, which can promote homologous recombination between DNA molecules.

[0146] As used herein, the term "mutated sequence" refers to a sequence that has undergone a sequence change compared to an unedited sequence. The mutant sequence includes a mutation at a single amino acid site or nucleotide site or multiple amino acid sites or nucleotide sites, or a mutation in one or more consecutive amino acid or nucleotide sequences. The mutation includes sequence deletion, sequence substitution, sequence insertion, etc. and any combination of these changes.

[0147] The term "donor DNA" refers to a DNA that can undergo homologous recombination with the DNA around the target region. The donor DNA includes a base sequence upstream of the target region and a base sequence downstream (e.g., a base sequence adjacent to the target region) as a homology arm. In this specification, the homology arm consisting of the base sequence on the upstream side of the target region (e.g., a base sequence adjacent to the upstream side) is sometimes referred to as an "upstream homology arm", and the homology arm consisting of the base sequence on the downstream side of the target sequence (e.g., a base sequence adjacent to the downstream side) is referred to as a "downstream homology arm". In the donor DNA, a desired base sequence can be included between the upstream homology arm and the downstream homology arm. The lengths of the upstream homology arm and the downstream homology arm can be the same or different from each other.

[0148] The "upstream" of the target region refers to the DNA region located on the 5' side of the nucleotide chain serving as the reference in the double-stranded DNA of the target region. The "downstream" of the target region refers to the DNA located on the 3' side of the nucleotide chain serving as the reference. In this specification, when a reference genome sequence of a public database is used as the source of the target region sequence, the upstream and downstream are generally determined based on the reference genome sequence.

[0149] In this specification, the sequence identity (or homology) of base sequences or amino acid sequences refers to: two base sequences or amino acid sequences are introduced into the part equivalent to the insertion and deletion in a manner that the consistency of the corresponding base or amino acid reaches the maximum degree and are arranged, and the value obtained in the form of the ratio of the consistent base or amino acid other than the interval in the obtained comparison relative to the whole base sequence or the whole amino acid sequence. The sequence identity of base sequences or amino acid sequences can be obtained using various homology search software known in the art. For example, the value of the sequence identity of the base sequence can be obtained by calculation based on the comparison obtained using the known homology search software BLASTN, and the value of the sequence identity of the amino acid sequence can be obtained by calculation based on the comparison obtained using the known homology search software BLASTP.

[0150] As used herein, the term "variant" is understood to be a molecule that is one or more changes in length or sequence compared to the molecule from which it is derived. The molecule from which the variant is derived is also referred to as a parent molecule. The term "variant" includes a "fragment" or "derivative" of a parent molecule. Generally, a "fragment" is smaller than a parent molecule in length or size, while a "derivative" shows one or more differences in its sequence compared to the parent molecule. Also included are modified molecules, such as, but not limited to, proteins (such as glycosylated, biotinylated, phosphorylated, ubiquitinated, palmitoylated or proteolytically cleaved proteins) and modified nucleic acids, such as methylated DNA, modified. The term "variant" also includes a mixture of different molecules, such as, but not limited to, RNA-DNA hybrids. Generally, variants are preferably artificially constructed by gene technology means, and the parent protein or polynucleotide is a wild-type protein or polynucleotide or its consensus sequence. However, naturally occurring variants are also understood to be included by the term "variant" as used herein. Furthermore, variants useful in the present invention may also be derived from homologs, orthologs or paralogs of a parent molecule or artificially constructed variants, as long as the variant exhibits at least one biological activity of the parent molecule, that is, is functionally active.

[0151] As used herein, the terms "protein sequence variant", "polypeptide variant" are understood to be a peptide, polypeptide or protein that differs in amino acid sequence by one or more changes compared to the protein or polypeptide from which it is derived. The peptide, polypeptide or protein from which the corresponding variant is derived is also referred to as the parent peptide, polypeptide or protein. Further, the variants that can be used in the present invention can also be derived from homologues, orthologues or lateral homologues of the parent peptide, polypeptide or protein, or artificially constructed variants, as long as the variant exhibits at least one biological activity of the parent peptide, polypeptide or protein. The changes in the amino acid sequence can be amino acid exchanges, insertions, deletions, N-terminal truncations or C-terminal truncations, or any combination of these changes, which can occur at one or several sites. A polypeptide or protein variant may exhibit a total of up to 200 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200) changes (i.e., exchanges, insertions, deletions, N-terminal truncations and / or C-terminal truncations) in the amino acid sequence. The amino acid exchanges may be conservative and / or non-conservative. On the other hand or in addition, a "variant" as used herein may be characterized by a certain degree of sequence identity with the parent peptide, polypeptide or protein from which it is derived. More precisely, a peptide, polypeptide or protein variant in the context of the present invention exhibits at least 50% sequence identity with its parent peptide, polypeptide or protein. The sequence identity of polypeptide or protein variants is over a contiguous stretch of 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids.

[0152] "Percent (%) amino acid sequence identity" with respect to a reference sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference parent sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of sequence identity for the sake of comparison. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. The FASTA program package is written by W.R. Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; W.R. Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36, and is publicly available from http: / / www.ebi.ac.uk / Tools / sss / fasta / . Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the sequences to be compared.

[0153] II. Specific implementation methods

[0154] In one aspect, the present disclosure provides a recombinant engineering system suitable for mycobacteria, which comprises an expression system of a gene sequence encoding a RecE protein or a homologue thereof and / or a RecT protein or a homologue thereof.

[0155] In some embodiments, the expression system comprises the gene sequence of optimized RecE protein and RecT protein. In some embodiments, the expression system comprises the gene sequence of optimized RecE protein or RecT protein. In some embodiments, the expression system comprises the gene sequence of optimized RecE protein homologue and RecT protein homologue. In some embodiments, the expression system comprises the gene sequence of optimized RecE protein homologue or RecT protein homologue.

[0156] In some embodiments, the RecE protein or a homolog thereof and the RecT protein or a homolog thereof are derived from mycobacteriophages, prophages or bacteria.

[0157] In some embodiments, the RecE protein or a homolog thereof and the RecT protein or a homolog thereof are from the same phage, prophage or bacterium.

[0158] In some embodiments, the RecE protein or a homolog thereof and the RecT protein or a homolog thereof are from different bacteriophages, prophages or bacteria.

[0159] In some embodiments, the expression system comprises only the gene sequence of the RecE protein or its homologue. In some embodiments, the RecE protein or its homologue is selected from the amino acid sequence shown in any one of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24 or any variant thereof.

[0160] In some embodiments, the expression system RecE protein or its homolog is selected from an amino acid sequence shown in any one of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, or a variant having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity with any of the aforementioned amino acid sequences.

[0161] In some embodiments, the expression system RecE protein or its homologue is selected from the amino acid sequence shown in any one of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, or a variant having at least 80%, 85%, 90%, 95% or 99% sequence identity with any of the aforementioned amino acid sequences. In some preferred embodiments, the expression system RecE protein is selected from the amino acid sequence shown in any one of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, or a variant having at least 80% sequence identity with any of the aforementioned amino acid sequences. In some preferred embodiments, the RecE protein or its homologue is selected from the amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 18, 20, 22, 24.

[0162] In some embodiments, the expression system comprises only the gene sequence of the RecT protein or its homologue. In some embodiments, the RecT protein or its homologue is selected from the amino acid sequence shown in any one of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25 or any variant thereof.

[0163] In some embodiments, the RecT protein or its homolog is selected from an amino acid sequence as shown in any one of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, or a variant having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity with any of the aforementioned amino acid sequences.

[0164] In some embodiments, the RecT protein or its homologue is selected from the amino acid sequence as shown in any one of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, or a variant having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity with any of the aforementioned amino acid sequences. In some preferred embodiments, the RecT protein is selected from the amino acid sequence as shown in any one of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, or a variant having at least 64% sequence identity with any of the aforementioned amino acid sequences. In some preferred embodiments, the RecT protein or its homologue is selected from the amino acid sequence having 64% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 19, 21, 23, 25.

[0165] In some embodiments, the RecT protein has an amino acid sequence as shown in SEQ ID NO: 19. In some embodiments, the RecT protein is encoded by a nucleotide sequence as shown in SEQ ID NO:33.

[0166] In some embodiments, the expression system comprises a combination of RecE protein or its homologue and RecT protein or its homologue. In some embodiments, the RecE protein or its homologue and RecT protein or its homologue are selected from any one of the following combinations:

[0167] (1) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 12 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 13 or any variant thereof;

[0168] (2) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 14 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 15 or any variant thereof;

[0169] (3) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 16 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 17 or any variant thereof;

[0170] (4) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 18 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 19 or any variant thereof;

[0171] (5) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 20 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 21 or any variant thereof;

[0172] (6) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 22 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 23 or any variant thereof;

[0173] (7) A RecE protein having an amino acid sequence as shown in SEQ ID NO: 24 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 25 or any variant thereof.

[0174] In some preferred embodiments, the RecE protein or its homologue and the RecT protein or its homologue are selected from any one of the following combinations:

[0175] (1) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 12, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 13;

[0176] (2) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 14, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 15;

[0177] (3) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 16, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 17;

[0178] (4) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 18, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 19;

[0179] (5) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 20 or a variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 21 or a variant thereof;

[0180] (6) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 22 or a variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 23 or a variant thereof;

[0181] (7) A RecE protein having an amino acid sequence as shown in SEQ ID NO: 24 or a variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 25 or a variant thereof.

[0182] In some preferred embodiments, the RecE protein or its homologue and / or the RecT protein or its homologue is selected from any one of the following combinations:

[0183] (1) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 18 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 19 or any variant thereof;

[0184] (2) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 20 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 21 or any variant thereof;

[0185] (3) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 22 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 23 or any variant thereof;

[0186] (4) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 24 or any variant thereof, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 25 or any variant thereof;

[0187] (5) A RecT protein having an amino acid sequence as shown in SEQ ID NO: 19 or any variant thereof.

[0188] In some preferred embodiments, the RecE protein or its homologue and / or the RecT protein or its homologue is selected from any one of the following combinations:

[0189] (1) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 18, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 19;

[0190] (2) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 20, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 21;

[0191] (3) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 22, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 23;

[0192] (4) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 24, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 25;

[0193] (5) A RecT protein having the amino acid sequence shown in SEQ ID NO: 19.

[0194] In some preferred embodiments, the RecE protein or its homologue and the RecT protein or its homologue are selected from any one of the following combinations:

[0195] (1) a RecE protein encoded by the nucleotide sequence shown in SEQ ID NO: 32, and a RecT protein encoded by the nucleotide sequence shown in SEQ ID NO: 33;

[0196] (2) a RecE protein encoded by the nucleotide sequence shown in SEQ ID NO: 34, and a RecT protein encoded by the nucleotide sequence shown in SEQ ID NO: 35;

[0197] (3) a RecE protein encoded by the nucleotide sequence shown in SEQ ID NO: 36, and a RecT protein encoded by the nucleotide sequence shown in SEQ ID NO: 37;

[0198] (4) The RecE protein encoded by the nucleotide sequence shown in SEQ ID NO:38, and the RecT protein encoded by the nucleotide sequence shown in SEQ ID NO:39.

[0199] In some embodiments, the expression system is integrated directly into the host genome.

[0200] In some embodiments, the expression system is encoded by a mobile genetic element. In some embodiments, the mobile genetic element is a plasmid, a transposon or a bacteriophage. In some preferred embodiments, the mobile genetic element is a plasmid. In some embodiments, the plasmid further comprises a promoter.

[0201] In some embodiments, the promoter is an inducible promoter or a constitutive promoter.

[0202] In some embodiments, the inducible promoter is a tetracycline-inducible promoter, a nitrile-inducible promoter, a thiostrepton-inducible promoter, or an acetamide-inducible promoter.

[0203] In some embodiments, the constitutive promoter is an hsp60 promoter, a pms promoter, a trp promoter, a Psmyc promoter, or a Pimyc promoter.

[0204] In some embodiments, recE gene and recT gene are transcribed from promoter separately respectively.In some embodiments, these two genes are organized in a cis operon, and its transcription is driven by the same promoter.The order of gene in the operon may affect the gene expression level.In some embodiments, recE gene is at recT gene upstream, and in some embodiments, recT gene is at recE gene upstream.

[0205] In some embodiments, the plasmid comprises a nucleotide sequence selected from any one of SEQ ID NOs: 40-59.

[0206] In some embodiments, the plasmid comprises a nucleotide sequence selected from any one of SEQ ID NOs: 49-59.

[0207] In some embodiments, the recombineering system further comprises a first recombination donor, which comprises an upstream homology arm and a downstream homology arm from the 5' end to the 3' end, respectively; optionally, a mutation sequence is further comprised between the upstream homology arm and the downstream homology arm, and the mutation sequence is used to replace the gene editing target sequence of the mycobacterium.

[0208] In some embodiments, the first recombination donor is double-stranded DNA or single-stranded DNA.

[0209] In some embodiments, the first recombinant donor further comprises a sequence homologous to the upstream homology arm, located between the upstream homology arm and the downstream homology arm. In some embodiments, the first recombinant donor further comprises a cleavage site for at least one DNA cleavage system, located between the upstream homology arm and the sequence homologous to the upstream homology arm.

[0210] In some embodiments, the first recombinant donor also comprises a sequence homologous to the downstream homology arm, between the upstream homology arm and the downstream homology arm. In some embodiments, the first recombinant donor also comprises a cleavage site of at least one DNA cutting system, between the downstream homology arm and the sequence homologous to the downstream homology arm.

[0211] In some embodiments, the first recombinant donor further comprises a sequence homologous to a fragment located upstream of the upstream homology arm binding region in the target molecule, which is located between the upstream homology arm and the downstream homology arm. In some embodiments, the first recombinant donor further comprises a cleavage site for at least one DNA cleavage system, which is located between the upstream homology arm and the sequence homologous to a fragment located upstream of the upstream homology arm binding region in the target molecule.

[0212] In some embodiments, the first recombinant donor further comprises a sequence homologous to a fragment located downstream of the downstream homology arm binding region in the target molecule, which is located between the upstream homology arm and the downstream homology arm. In some embodiments, the first recombinant donor further comprises a cleavage site for at least one DNA cleavage system, which is located between the downstream homology arm and the sequence homologous to a fragment located downstream of the downstream homology arm binding region in the target molecule.

[0213] In another aspect, the present disclosure provides a tool kit for scarless genome editing, comprising the aforementioned recombineering system, and optionally a DNA cleavage system.

[0214] In some embodiments, the DNA cleavage system is selected from a homing endonuclease, a CRISPR-Cas system, a transcription activator-like effector nuclease (TALENs) or a zinc finger nuclease (ZFNs). In some embodiments, the homing endonuclease is I-SceI.

[0215] In some embodiments, the tool box further comprises a second recombination donor for guiding DNA repair. In some embodiments, the second recombination donor comprises an upstream homology arm and a downstream homology arm from the 5' end to the 3' end, respectively. In some embodiments, the second recombination donor further comprises a mutant sequence located in the middle of the upstream homology arm and the downstream homology arm.

[0216] In another aspect, the present disclosure provides a genetically engineered bacterium comprising the aforementioned recombinant engineering system or the aforementioned tool kit.

[0217] In another aspect, the present disclosure provides a method for performing scarless genome editing using the aforementioned composition, comprising the following steps:

[0218] Providing a host cell, wherein the host cell comprises the aforementioned recombineering system or the aforementioned tool kit;

[0219] transforming the first recombinant donor into a host cell;

[0220] Using a recombineering system comprising RecE protein or its homolog and / or RecT protein or its homolog to mediate the recombination of the first recombination donor and the gene editing target molecule;

[0221] Optionally, the method further comprises the steps of:

[0222] Targeted DNA cleavage using a DNA cleavage system.

[0223] The first recombinant donor or the second recombinant donor is used to guide DNA repair.

[0224] In a preferred embodiment, the scarless gene editing method is achieved by the combined action of a recombineering system comprising RecE protein and / or RecT protein and a DNA cutting system. In a preferred embodiment, the scarless gene editing is carried out in three steps, the first step is the first recombinant donor (including upstream homology arms, downstream homology arms and mutant sequences therebetween), mediated by RecE / RecT, and recombined with the target molecule. The second step is the cutting of the target molecule cutting site catalyzed by the DNA cutting system. The third step is DNA repair guided by the first donor or the second donor.

[0225] In some embodiments, in the first step, the first recombinant donor includes at least one cleavage site between the upstream homology arm and the downstream homology arm, which is recognized by the DNA cutting system. In some embodiments, the first recombinant donor includes more than one cleavage site, located between the upstream homology arm and the downstream homology arm. In some embodiments, the first recombinant donor includes a mutant sequence intended for knock-in, located between the upstream homology arm and the cleavage site, or / and between the downstream homology arm and the cleavage site. In some embodiments, the first recombinant donor also includes a sequence homologous to the upstream homology arm, located between the upstream homology arm and the downstream homology arm, and there is at least one cleavage site between the upstream homology arm and the sequence homologous to the upstream homology arm. In some embodiments, the first recombinant donor also includes a sequence homologous to the downstream homology arm, located between the upstream homology arm and the downstream homology arm, and there is at least one cleavage site between the downstream homology arm and the sequence homologous to the downstream homology arm. In some embodiments, the first recombinant donor also includes a sequence homologous to a fragment located upstream of the upstream homology arm binding region in the target molecule, located between the upstream homology arm and the downstream homology arm, and there is at least one cleavage site between the upstream homology arm and the sequence homologous to a fragment located upstream of the upstream homology arm binding region in the target molecule. In some embodiments, the first recombinant donor also includes a sequence homologous to a fragment located downstream of the downstream homology arm binding region in the target molecule, located between the upstream homology arm and the downstream homology arm, and there is at least one cleavage site between the downstream homology arm and the sequence homologous to a fragment located downstream of the downstream homology arm binding region in the target molecule. In a specific embodiment, the first recombinant donor includes an upstream homology arm, a sequence intended for knock-in, a sequence homologous to the downstream homology arm, a cleavage site, an antibiotic marker, a cleavage site, and a downstream homology arm in sequence from the 5' to the 3' end. In a specific embodiment, the first recombinant donor includes, from 5' to 3' end, an upstream homology arm, a sequence intended for knock-in, a sequence homologous to a fragment located downstream of the downstream homology arm binding region in the target molecule, a cleavage site, an antibiotic marker, a cleavage site, and a downstream homology arm. In a specific embodiment, the first recombinant donor includes, from 5' to 3' end, an upstream homology arm, a cleavage site, an antibiotic marker, a cleavage site, a sequence homologous to the upstream homology arm, a sequence intended for knock-in, and a downstream homology arm. In a specific embodiment, the first recombinant donor includes, from 5' to 3' end, an upstream homology arm, a cleavage site, an antibiotic marker, a cleavage site, a sequence homologous to a fragment located upstream of the upstream homology arm binding region in the target molecule, a sequence intended for knock-in, and a downstream homology arm.

[0226] In some embodiments, in the second step, the DNA cutting system is encoded by a mobile gene element, such as a plasmid. In certain embodiments, the DNA cutting system is located on the same mobile gene element as the RecE / RecT system. In certain embodiments, the DNA cutting system is integrated into the host genome. The DNA cutting system can be constitutively expressed or induced to express. In certain embodiments, the DNA cutting system is selected from a homing endonuclease, a CRISPR-Cas system, a transcription activator-like effector nuclease (TALENs) or a zinc finger nuclease (ZFNs). In certain embodiments, the DNA cutting system is a CRISPR-Cas system. In certain embodiments, the DNA cutting system is a homing endonuclease.

[0227] In some embodiments, in the third step, a second donor molecule including an upstream homology arm and a downstream homology arm is provided to guide DNA repair. In some embodiments, the second donor also includes a mutant sequence intended for knock-in between the upstream homology arm and the downstream homology arm. In some embodiments, a second donor is not provided, and DNA repair occurs by the recombination between the sequence homologous to the upstream homology arm and the upstream homology arm of the first donor that has been integrated into the target molecule. In some embodiments, a second donor is not provided, and DNA repair occurs by the recombination between the sequence homologous to the downstream homology arm and the downstream homology arm of the first donor that has been integrated into the target molecule. In some embodiments, a second donor is not provided, and DNA repair occurs by the recombination between the sequence homologous to the downstream homology arm and the downstream homology arm of the first donor that has been integrated into the target molecule. In some embodiments, a second donor is not provided, and DNA repair occurs by the recombination between the sequence homologous to a fragment located upstream / downstream of the upstream / downstream homology arm binding region in the target molecule encoded by the first donor in the above-mentioned first step and the homologous sequence in the target molecule.

[0228] In some embodiments, the method comprises the following steps:

[0229] Providing a host cell, wherein the host cell comprises the aforementioned recombineering system or the aforementioned tool kit;

[0230] Transforming a first recombinant donor into a host cell, wherein the first recombinant donor comprises, from the 5' end to the 3' end, an upstream homology arm, optionally a mutation sequence, a downstream homology arm, and at least one cleavage site located between the upstream homology arm and the downstream homology arm, respectively, and the cleavage site is recognized by a DNA cleavage system;

[0231] Using a recombineering system comprising RecE protein and / or RecT protein to mediate the recombination of the first recombinant donor and the gene editing target molecule;

[0232] Utilize DNA cleavage system for targeted DNA cleavage;

[0233] The second recombinant donor is used to guide DNA repair, wherein the second recombinant donor comprises an upstream homology arm and a downstream homology arm from the 5' end to the 3' end, respectively, and optionally, a mutation sequence located between the upstream homology arm and the downstream homology arm.

[0234] In some embodiments, the method comprises the following steps:

[0235] Providing a host cell, wherein the host cell comprises the aforementioned recombineering system or the aforementioned tool kit;

[0236] Transforming a first recombinant donor into a host cell, wherein the first recombinant donor comprises, from the 5' end to the 3' end, an upstream homology arm, optionally a mutant sequence, a downstream homology arm, and a sequence homologous to the upstream or downstream homology arm located between the upstream homology arm and the downstream homology arm, and at least one cleavage site located between the upstream or downstream homology arm and the sequence homologous to the upstream or downstream homology arm, and the cleavage site is recognized by a DNA cleavage system;

[0237] Using a recombineering system comprising RecE protein and / or RecT protein to mediate the recombination of the first recombinant donor and the gene editing target molecule;

[0238] Utilize DNA cleavage system for targeted DNA cleavage;

[0239] DNA repair is directed by recombination between homologous sequences in the first recombination donor and the upstream or downstream homology arms.

[0240] In some embodiments, the method comprises the following steps:

[0241] Providing a host cell, wherein the host cell comprises the aforementioned recombineering system or the aforementioned tool kit;

[0242] Transforming a first recombinant donor into a host cell, wherein the first recombinant donor comprises, from the 5' end to the 3' end, an upstream homology arm, an optionally mutated sequence, a downstream homology arm, and a sequence between the upstream homology arm and the downstream homology arm homologous to a fragment of the target molecule located upstream of the upstream homology arm binding region, and at least one cleavage site between the upstream homology arm and the sequence homologous to a fragment of the target molecule located upstream of the upstream homology arm binding region, or a sequence between the upstream homology arm and the downstream homology arm homologous to a fragment of the target molecule located downstream of the downstream homology arm binding region, and at least one cleavage site between the downstream homology arm and the sequence homologous to a fragment of the target molecule located downstream of the downstream homology arm binding region, and the cleavage site is recognized by a DNA cleavage system;

[0243] Using a recombineering system comprising RecE protein and / or RecT protein to mediate the recombination of the first recombinant donor and the gene editing target molecule;

[0244] Utilize DNA cleavage system for targeted DNA cleavage;

[0245] DNA repair is guided by recombination between homologous sequences in the first recombinant donor and corresponding homologous sequences upstream or downstream in the target molecule.

[0246] In some embodiments, the DNA cleavage system is selected from a homing endonuclease, a CRISPR-Cas system, a transcription activator-like effector nuclease (TALENs), or a zinc finger nuclease (ZFNs).

[0247] In some embodiments, the homing endonuclease is I-Scel.

[0248] In some embodiments, the host cell is a mycobacterium microorganism. In some embodiments, the mycobacterium is a fast-growing mycobacterium, including Mycobacterium smegmatis, Mycobacterium fortuitum, Mycobacterium flavosum, and Mycobacterium neogoldenum. In some embodiments, the mycobacterium is a slow-growing mycobacterium, including Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium complex, Mycobacterium surga, Mycobacterium malmo, Mycobacterium scrofula, and Mycobacterium scaly. In some embodiments, the mycobacterium is Mycobacterium neogoldenum. In some embodiments, the mycobacterium is M.neoaurum strain NRRL B-3805.

[0249] Example

[0250] A further understanding of the present disclosure can be obtained by referring to some specific embodiments given herein, which are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. Obviously, various modifications and changes can be made to the present disclosure without departing from the essence of the present disclosure, and therefore, these modifications and changes are also within the scope of the protection required by the present application.

[0251] Example 1. Testing the gene replacement rate mediated by pJV53

[0252] M. neoaurum strain NRRL B-3805 was purchased from ARS Culture Collection (NRRL) ( https: / / nrrl.ncaur.usda.gov / ). The public database genome sequence GenBank: CP011022.1 was used as the reference sequence of the B-3805 strain genome. The gene MyAD_23825 in the B-3805 genome was selected for gene replacement research. The specific plan is: design a linear dsDNA donor and replace the MyAD_23825 gene fragment with a gentamicin resistance box. Donor Donor I (SEQID NO: 4) contains an upstream homology arm (SEQ ID NO: 1), a gentamicin resistance box (SEQ ID NO: 2) and a downstream homology arm (SEQ ID NO: 3) from the 5' end to the 3' end. The schematic diagram of the expected product of gene replacement is shown in Figure 2 As shown. Plasmid pJV53 was purchased from lifescience-market ( https: / / www.lifescience-market.com / plasmid-c-94 / pjv53-p- 107938.html) .

[0253] The plasmid was transformed into the B-3805 strain according to the protocol described by Loraine and Smith (2017). The transformant strain was named S1.

[0254] The transcription of the Che9c gp60 and gp61 genes encoded by pJV53 is controlled by an acetamide-inducible promoter. To investigate whether the induction of gp60 and gp61 expression has an effect on the efficiency of gene replacement, two types of competent cells of the S1 strain were prepared: uninduced and induced. For uninduced competent cells, the protocol described by Loraine and Smith (2017) was followed. For induced competent cells, the protocol described by Loraine and Smith (2017) was followed with one adjustment, namely, acetamide was added to the culture approximately 4 hours before cell harvest, the inducer was added at a concentration of 34 mM, and then the culture was continued for 4 hr.

[0255] Uninduced and induced S1 competent cells were transformed with 500ng Donor I molecules. The transformant culture was plated on a solid medium containing 50mg / L kanamycin sulfate and 50mg / L gentamicin sulfate and cultured in a 30°C incubator for 5-8 days. 42 and 45 transformant colonies were obtained from the transformation of uninduced competent cells and induced competent cells, respectively. As a negative control for transformation, competent cells that were not transformed with Donor I were also plated on a selective solid medium, and no colonies grew.

[0256] The inventors designed relevant primers to verify whether the transformant colonies grown on selective solid medium contain the expected gene replacement, such as Figure 2As shown. Primer P1 (SEQ ID NO: 5) binds to the genomic sequence upstream of the upstream homology arm binding region, and primer P2 (SEQ ID NO: 6) binds to the gentamicin resistance box sequence. This pair of primers is used to verify whether the sequence around the upstream homology arm binding region is consistent with expectations. Primer P3 (SEQ ID NO: 7) binds to the genomic sequence downstream of the downstream homology arm binding region, and primer P4 (SEQ ID NO: 8) binds to the gentamicin resistance box sequence. This pair of primers is used to verify whether the sequence around the downstream homology arm binding region is consistent with expectations. Gene replacement is confirmed only when both PCR reactions (P1+P2 and P3+P4) produce the expected amplification products (1.8kb and 1.7kb, respectively).

[0257] Twenty-four colonies of each transformant obtained from the uninduced and induced competent cells were tested by PCR. The results are shown in Figure 3 .Depend on Figure 3 It can be seen that most colonies only produce PCR products of the expected size around the upstream homology arm or the downstream homology arm, but not in both detection areas. This is obviously inconsistent with the expected results of gene replacement. Although the gene replacement efficiency mediated by pJV53 is reported in the literature to be very high (>90%) (van Kessel et al. 2007), our data show that one or some undesirable recombination events dominate, and the recombination results are different from the expected products of gene replacement. Among the transformant colonies tested, only 3 transformant colonies under uninduced conditions and 2 transformant colonies under induced conditions showed the expected PCR products in both the upstream homology arm and the downstream homology arm regions, which are consistent with the expected gene replacement products. Therefore, the actual gene replacement efficiency is calculated to be 12.5% ​​(3 / 24) and 8% (2 / 24).

[0258] There were no significant differences between induced and uninduced conditions, which may be due to leaky expression from the acetamide-inducible promoter under uninduced conditions.

[0259] Example 2. Establishment of a convenient and effective method for detecting gene replacement efficiency

[0260] In order to conveniently determine the gene replacement efficiency, a green fluorescent protein (GFP) expression strain was constructed by integrating a GFP expression cassette (SEQ ID NO: 9) into the non-coding region between the MyAD_02345 gene and the MyAD_02355 gene in the B-3805 genome. See the schematic diagram. Figure 4 In subsequent gene replacement experiments, the GFP gene will be used as the target molecule, and the results can be observed by the disappearance of the fluorescent phenotype.

[0261] The GFP expression cassette was integrated into the genome using a suicide plasmid (integration plasmid). Figure 5 The plasmid was synthesized by Beijing Liuhe BGI Technology Co., Ltd.

[0262] The genome was modified by suicide plasmid (see Kendall and Frita, 2009), and the resulting double-crossover strain (i.e., a strain containing a GFP expression cassette at a designated genomic site) was named S2. S2 showed a green fluorescent phenotype under a blue light source and did not grow on SOB solid medium containing 50 mg / L gentamicin sulfate.

[0263] Example 3. Construction and testing of a recombineering system encoding a novel RecE / RecT protein

[0264] Some novel RecE / RecT-like proteins derived from mycobacteriophages, prophages or mycobacterium microorganisms were obtained through bioinformatics analysis. These proteins were codon optimized to obtain the corresponding gene sequences. The protein sequence and gene sequence of the novel RecE / RecT-like proteins are shown in Table 1.

[0265] Table 1. RecE / RecT-like proteins studied in this disclosure

[0266] protein Amino Acid Sequence ID Nucleotide sequence ID RecE2 SEQ ID NO:12 SEQ ID NO:26 RecT2 SEQ ID NO:13 SEQ ID NO:27 RecE4 SEQ ID NO:14 SEQ ID NO:28 RecT4 SEQ ID NO:15 SEQ ID NO:29 RecE5 SEQ ID NO:16 SEQ ID NO:30 RecT5 SEQ ID NO:17 SEQ ID NO:31 RecE6 SEQ ID NO:18 SEQ ID NO:32 RecT6 SEQ ID NO:19 SEQ ID NO:33 RecE8 SEQ ID NO:20 SEQ ID NO:34 RecT8 SEQ ID NO:21 SEQ ID NO:35 RecE9 SEQ ID NO:22 SEQ ID NO:36 RecT9 SEQ ID NO:23 SEQ ID NO:37 RecE10 SEQ ID NO:24 SEQ ID NO:38 RecT10 SEQ ID NO:25 SEQ ID NO:39

[0267] Use the Clustal Omega web tool ( https: / / www.ebi.ac.uk / jdispatcher / msa / cluster ) The sequence alignment analysis of the novel RecE-like proteins was performed, and their protein sequence identities with Che9c gp60 (Uniprot ID: Q854U0) are shown in Table 2:

[0268] Table 2. Sequence identity percentage matrix of RecE-like protein and Che9c gp60 protein

[0269]

[0270]

[0271] Use the Clustal Omega web tool ( https: / / www.ebi.ac.uk / jdispatcher / msa / cluster) The sequence alignment analysis of the novel RecT-like proteins was performed, and their protein sequence identities with Che9c gp61 (Uniprot ID: Q854T9) and Halo gp43 (Uniprot ID: Q1A0N6) are shown in Table 3:

[0272] Table 3. Sequence identity percentage matrix of RecT-like proteins with Che9c gp61 and Halo gp43 proteins

[0273]

[0274] The new recombineering system includes plasmids expressing the RecE / RecT genes listed in Table 1 in pairs or expressing only a single RecT gene. The plasmids were synthesized by Beijing Liuhe BGI Technology Co., Ltd. These plasmids (see Table 4) were transformed into the S2 strain to create multiple strains containing the new RecE / RecT system.

[0275] Since some plasmids contain tetracycline-inducible promoters, two types of competent cells were prepared for transformant strains containing these plasmids: uninduced and induced (induced by adding 0.5 mg / L of dehydrated tetracycline to the culture 4 hours before competent cell harvest, similar to the description in Example 1). Some plasmids contain constitutive promoters, so only uninduced competent cells were prepared.

[0276] The experimental design of gene replacement in this example is to replace a sequence in the GFP gene coding sequence in the S2 genome with a gentamicin resistance box, so that the GFP gene is destroyed. Donor II (SEQ ID NO: 11) targeting the GFP gene includes a 500 bp upstream homology arm, a gentamicin resistance box and a 500 bp downstream homology arm from the 5' end to the 3' end.

[0277] These strains containing the novel RecE / RecT system were transformed with Donor II. The gene replacement efficiency was defined as the percentage of non-fluorescent colonies in the total colonies. The sequence IDs, main element descriptions, and experimentally obtained gene replacement efficiencies of the above plasmids are listed in Table 4.

[0278] Table 4 Plasmids encoding novel RecE / RecT proteins and the gene replacement rates mediated by them.

[0279]

[0280]

[0281] From the above results, it can be seen that some recombineering systems encoding novel RecE / RecT proteins showed a gene replacement rate of >90%, and some even reached a gene replacement rate of 100%. Most of these systems contain both RecE and RecT proteins, but there are also examples of high gene replacement rates in systems containing only RecT proteins, such as pBotaET22. Among the plasmids containing inducible promoters, some plasmids showed the best gene replacement efficiency under induced conditions, such as pBotaET21; some plasmids showed the best gene replacement efficiency under uninduced conditions, such as pBotaET20, pBotaET26, pBotaET27, pBotaET28, pBotaET35, pBotaET36; and some plasmids showed high gene replacement efficiency under both induced and uninduced conditions, such as pBotaET22.

[0282] As can be seen from Table 2, the sequence identities between RecE proteins (RecE6, RecE8, RecE9, RecE10) in the recombineering system with a higher gene replacement rate and Che9c gp60 are all less than 17%, but the sequence identity between them is above 80%.

[0283] Similarly, as can be seen from Table 3, the sequence identities between RecT proteins (RecT6, RecT8, RecT9, RecT10) in the recombineering system with a higher gene replacement rate and Che9c gp61 are all less than 37%, and the sequence identity between them and Halo gp43 is even less than 20%, but the sequence identity between them is above 64%.

[0284] Example 4. Evaluation of the efficiency of the new recombineering system using ssDNA donors

[0285] The experimental design of this example is to introduce A128->G nucleotide mutation in the gene MyAD_06390 encoding ribosomal protein S12 in the genome using ssDNA donors. This nucleotide point mutation will lead to K43->R amino acid point mutation in the S12 protein. It is known that the S12 protein with this point mutation will make mycobacteria acquire streptomycin resistance (Nair et al., Al. 1993), so the mutant product will be screened by the streptomycin resistance phenotype.

[0286] pBotaET26 was chosen as the representative RecE / RecT system for this and subsequent examples because it mediates gene replacement with high efficiency (97.7%) under uninduced conditions (Example 3). pBotaET26 was transformed into the S2 strain to obtain the S3 strain.

[0287] The ssDNA donor (Donor XXIII, SEQ ID NO: 60) includes an upstream homology arm and a downstream homology arm, both of which are 45 nt in length. The A->G mutation to be introduced is located between the upstream homology arm and the downstream homology arm of donor XXIII.

[0288] Use 5 pmole of Donor XXIII to transform the S3 strain (uninduced). Spread a portion of the transformant culture on a solid medium containing 100 mg / L streptomycin sulfate and culture in a 30°C incubator for 5-8 days. The culture results are shown in Figure 6 , hundreds of transformant colonies grew on the selective medium. As a negative control, S3 competent cells of untransformed Donor XXIII were also plated on solid medium containing 100 mg / L streptomycin sulfate, and no colonies grew on the selective medium.

[0289] Twelve streptomycin-resistant transformant colonies on selective medium were randomly selected to verify the introduced mutation. The sequence around the target site in the transformant genome was amplified by PCR using primers P9 (SEQ ID NO: 61) and P10 (SEQ ID NO: 62). The PCR product was subjected to Sanger sequencing using primers P9 and P10. The sequencing results were compared with the parent gene sequence. Figure 7 All transformants contained the expected A128->G mutation, indicating that the new RecE / RecT system can also efficiently mediate point mutations in the genome using ssDNA donors.

[0290] Example 5. Scarless genome editing method combining a novel RecE / RecT recombineering system and a DNA cleavage system

[0291] 5.1 Genomic integration of the first donor containing the homing endonuclease cleavage site

[0292] Schematic diagram of scarless gene editing using the RecE / RecT system is shown in Figure 1 .

[0293] The experimental design of this example is to replace the GFP expression cassette in the genome of strain S3 with a gentamicin resistance cassette, which has an I-SceI homing endonuclease cleavage site on each side. This is the first step of scarless genome editing. The first donor Donor XXIV (SEQ ID NO: 63) contains an upstream homology arm (SEQ ID NO: 64), an I-SceI cleavage site, a gentamicin resistance cassette, a second I-SceI cleavage site and a downstream homology arm (SEQ ID NO: 65) from the 5' end to the 3' end.

[0294] 500 ng of donor XXIV was transformed into S3 competent cells (uninduced). The transformant culture was plated on a solid medium containing 50 mg / L kanamycin sulfate and 50 mg / L gentamicin sulfate and cultured in a 30°C incubator for 5-8 days. The obtained colonies were almost all non-fluorescent transformants.

[0295] The non-fluorescent transformant strain with donor XXIV integrated into the genome was named S7.

[0296] 5.2 Construction of plasmid encoding I-SceI homing endonuclease and second donor

[0297] After the first donor (donor XXIV) is integrated into the genome, in order to achieve scarless genome editing, the gentamicin resistance cassette needs to be removed by cleavage with the I-SceI homing endonuclease (the second step of scarless genome editing, Figure 1 At the same time, a second donor is provided to guide DNA repair to ultimately achieve scarless genome editing (the third step of scarless genome editing, Figure 1 ). Therefore, it is necessary to introduce a second plasmid encoding the I-SceI homing endonuclease and the second donor into the S7 strain.

[0298] The I-SceI homing endonuclease sequence was downloaded from the UniProt website (UniProt ID: P03882). The nucleotide sequence of the I-SceI gene (SEQ ID NO: 66) was generated using the codon optimization tool on the IDT website.

[0299] For compatibility with the pBotaET26 plasmid in the S7 strain, the second plasmid utilized the replicon sequence of the compatible plasmid pMF1 (GenBank: AJ238973.1, Bachrach et al., 2000).

[0300] Two second donors were designed to guide DNA repair after I-SceI cleavage: one for scarless deletion (donor XXV), whose expected recombination product is the deletion of the gentamicin resistance cassette and the I-SceI cleavage sites on both sides of the genome of the S7 strain, that is, compared with the ancestral S3 strain, the GFP expression cassette in its genome is scarlessly deleted; the other for scarless replacement (donor XXVI), whose expected recombination product is the replacement of the gentamicin resistance cassette and the I-SceI cleavage sites on both sides of the genome of the S7 strain with a red fluorescent protein (RFP) expression cassette, that is, compared with the ancestral S3 strain, the GFP expression cassette is scarlessly replaced by the RFP expression cassette. Donor XXV (SEQ ID NO: 67) contains upstream homology arms and downstream homology arms from the 5' end to the 3' end. Donor XXVI (SEQID NO: 68) not only contains the same upstream homology arms and downstream homology arms as donor XXV, but also contains an RFP expression cassette (SEQ ID NO: 69) between the upstream and downstream homology arms.

[0301] The second plasmid pBotaET37 (SEQ ID NO: 70, map as shown in Figure 8 The plasmid comprises a replicon of pBR322, a replicon of pMF1, an apramycin resistance cassette, a donor XXV (SEQ ID NO: 67) and an I-SceI expression cassette.

[0302] Another second plasmid pBotaET38 (SEQ ID NO: 71, map as shown Fig. 9 The plasmid comprises a pBR322 replicon, a pMF1 replicon, an apramycin resistance cassette, a donor XXVI (SEQ ID NO: 68) and an I-SceI expression cassette. The donor XXVI comprises an RFP expression cassette.

[0303] 5.3 Scarless gene editing

[0304] S7 (uninduced) was transformed with the second plasmids pBotaET37 and pBotaET38, respectively. The transformant culture was plated on solid medium containing 50 mg / L kanamycin sulfate and 50 mg / L apramycin sulfate.

[0305] Schematic diagram of the target region in the S7 genome and the expected editing products mediated by pBotaET37 and pBotaET38 are shown in Fig.11 .

[0306] Eight transformant colonies from each transformation reaction were randomly selected and inoculated into YPD liquid medium (Yeast Extract Peptone Dextrose Medium), and cultured in a shaking incubator at 30°C for 3 days. Fig.10 From the obtained bacterial solution, all pBotaET37 transformants were non-fluorescent, while 7 pBotaET38 transformants showed red fluorescence.

[0307] These transformant strains were further tested by PCR. One PCR reaction used primers P11 (SEQ ID NO: 72) and P12 (SEQ ID NO: 73), which bind to the sequence in the genome that recombined with the second donor on both sides. When the template used in the PCR reaction was the same genotype as the parent strain S7, this pair of primers produced a 2.1 kb product; when the transformant genome contained the expected DNA modification, the pBotaET37 transformant strain produced a 1.1 kb product and the pBotaET38 transformant strain produced a 2 kb product. This PCR reaction was suitable for testing pBotaET37 transformants, but the product of pBotaET38 transformants was similar in size to the product of the parent strain and could not be distinguished. For pBotaET38 transformants, another PCR reaction was used with primers P11 (SEQ ID NO: 72) and primer P13 (SEQ ID NO: 74) that binds to the RFP expression cassette. When the pBotaET38 transformant contained the expected DNA modification, P11 and P13 produced a 1 kb product. The electrophoresis results of the PCR products were as follows: Fig.12 shown.

[0308] Depend on Fig.12 A and Fig.12 B As can be seen, in the PCR reaction using P11 and P12 primers, 7 out of 8 pBotaET37 transformants produced the expected PCR product (1.1 kb), confirming the deletion of the gentamicin resistance cassette; while 7 out of 8 pBotaET38 transformants produced a product of about 2 kb, and it was impossible to distinguish whether it was the parent or the recombinant product based on the size of the product. In the PCR reaction using P11 and P13 primers, the above 7 pBotaET38 transformants all produced the expected PCR product (1 kb), confirming that the RFP expression cassette replaced the gentamicin resistance cassette. In this experiment, the deletion or replacement efficiency of the gentamicin resistance cassette was 87.5% (7 / 8).

[0309] To further confirm that the genome editing that occurred was scarless, two PCR products generated by primers P11 and P12 from pBotaET37 and pBotaET38 transformants that passed the PCR test were randomly selected and Sanger sequenced using primers P11 and P12. The sequencing results were compared with the sequence of the expected editing product. Fig.13 , it can be seen that there is no gentamicin cassette or I-SceI cutting site in the genome of the selected transformant, and the editing product sequence is consistent with the expected product sequence.

[0310] Example 6 Scarless genome editing method 2 combining the novel RecE / RecT recombination engineering system and DNA cleavage system

[0311] 6.1 Genomic integration of the first donor containing a homing endonuclease cleavage site and sequences homologous to the downstream homology arms

[0312] The experimental design of this embodiment is to replace the GFP expression cassette in the genome of strain S3 with a gentamicin resistance cassette, and there is an I-SceI homing endonuclease cleavage site on each side of the gentamicin resistance cassette. The first donor Donor XXVII (SEQ ID NO: 75) comprises an upstream homology arm (SEQ ID NO: 64), a sequence homologous to the downstream homology arm (SEQ ID NO: 76), an I-SceI cleavage site, a gentamicin resistance cassette, a second I-SceI cleavage site and a downstream homology arm (SEQ ID NO: 65) from the 5' end to the 3' end.

[0313] 500 ng of donor XXVII was used to transform S3 competent cells (uninduced). The transformant culture was plated on a solid medium containing 50 mg / L kanamycin sulfate and 50 mg / L gentamicin sulfate and cultured in a 30°C incubator for 5-8 days. The resulting colonies were almost all non-fluorescent transformants.

[0314] The non-fluorescent transformant strain with donor XXVII integrated into the genome was named S8.

[0315] 6.2 Construction of plasmid encoding I-SceI

[0316] In order to achieve scarless genome editing, after the genomic integration of donor XXVII, the gentamicin resistance cassette needs to be removed by cutting with I-SceI homing endonuclease. Recombination between the homologous sequence (SEQ ID NO: 76) encoded by the first donor Donor XXVII and the downstream homology arm (SEQ ID NO: 65) can guide DNA repair to achieve scarless gentamicin resistance cassette deletion without providing a second donor. Therefore, it is necessary to introduce a second plasmid encoding I-SceI homing endonuclease into the S8 strain.

[0317] The second plasmid pBotaET39 (SEQ ID NO: 77, map as shown in Fig.14 As shown), the plasmid includes the replicon of pBR322, the replicon sequence of pMF1, the apramycin resistance cassette and the I-SceI expression cassette.

[0318] 6.3 Scarless gene editing

[0319] S8 (uninduced) was transformed with pBotaET39. The transformant culture was plated on solid medium containing 50 mg / L kanamycin sulfate and 50 mg / L apramycin sulfate. A schematic diagram of the target region in the S8 genome and the expected product of pBotaET39-mediated genome editing is shown in Fig.15 .

[0320] Eight transformant colonies were randomly selected for PCR examination. Primers P14 (SEQ ID NO: 78) and P15 (SEQ ID NO: 79) bind to both sides of the sequence in the genome that recombined with the first donor. When the template used in the PCR reaction is the same as the genotype of the parent strain S8, this pair of primers produces a 1.67kb product; when the pBotaET39 transformant genome contains the expected DNA modification (i.e., the deletion of the gentamicin resistance cassette), a 626bp product ( Fig.15 ). The electrophoresis results of PCR products are as follows Fig.16 As shown, all 8 transformants produced a product of about 626 bp. That is, in this experiment, the deletion efficiency of the gentamicin resistance cassette was 100% (8 / 8).

[0321] To confirm that the genome editing that occurred was scarless, PCR products from five pBotaET39 transformants were randomly selected and Sanger sequenced using primers P14 and P15. The sequencing results were compared with the expected edited sequences. Fig.17 , it can be seen that there is no gentamicin resistance cassette or I-SceI cleavage site in the genome of the selected transformant, and the editing product sequence is consistent with the expected product sequence.

[0322] Example 7 Scarless genome editing method combining novel RecE / RecT recombineering system and DNA cleavage system

[0323] 7.1 Genomic integration of a first donor comprising a homing endonuclease cleavage site and a sequence homologous to a sequence in the genome located downstream of the downstream homology arm binding region

[0324] In order to prove that the genome editing tool developed by the present invention is not only applicable to the editing of a specific gene or site (such as a GFP expression box) in the genome, this example selects another target gene in the genome, the MyAD_23740 gene.

[0325] The experimental design of this embodiment is to replace the MyAD_23740 gene in the genome with a gentamicin resistance box, and there is an I-SceI homing endonuclease cleavage site on each side of the gentamicin resistance box. The first donor Donor XXVIII (SEQ ID NO: 80) includes an upstream homology arm (SEQ ID NO: 81) from the 5' end to the 3' end, a sequence homologous to a sequence located downstream of the downstream homology arm binding region in the genome (SEQ ID NO: 82), an I-SceI cleavage site, a gentamicin resistance box, a second I-SceI cleavage site and a downstream homology arm (SEQ ID NO: 83).

[0326] Strain S1 was transformed with pBotaET26, and the resulting transformant strain was named S10. 500 ng of donor XXVIII was used to transform S10 competent cells (uninduced), and the transformant culture was plated on a solid medium containing 50 mg / L kanamycin sulfate and 50 mg / L gentamicin sulfate and cultured in a 30°C incubator for 5-8 days. Since S10 has no fluorescent phenotype, whether the genomic integration of donor XXVIII occurs in the resulting transformant colonies needs to be detected by PCR.

[0327] The first PCR reaction used primers P16 (SEQ ID NO: 84) and P17 (SEQ ID NO: 85), which bind to both sides of the sequence in the genome that recombined with donor XXVIII. When the template used in the PCR reaction is the same genotype as the parent strain S10, this pair of primers produces a 1.9 kb product; when the transformant genome contains the expected donor integration, a 1.7 kb product is produced. However, the two PCR products are difficult to distinguish during electrophoresis because of their similar size. The second PCR reaction used primers P16 and P18 (SEQ ID NO: 86), where P18 is a primer specific for the donor sequence. When the transformant genome contains the expected donor integration, this pair of primers produces a 671 bp product. The third PCR reaction used primers P17 and P19 (SEQ ID NO: 87), where P19 is a primer specific for the donor sequence. When the transformant genome contains the expected donor integration, this pair of primers produces a 611 bp product. When all three PCR reactions produced products of the expected size, the transformants were confirmed to be the expected donor integration products. A schematic diagram of the target region in the S11 genome and the expected products of genome editing mediated by pBotaET39 is shown in Fig.18 .

[0328] Six transformant colonies were randomly selected for PCR detection. The results are shown in Fig.19 The results showed that these 6 transformants were all the expected integration products of Donor XXVIII, that is, the integration efficiency was 100% (6 / 6). This result further proves that the new RecE / RecT system in the present invention can achieve a high gene replacement rate at different genomic sites, and the recombination results are consistent with the expected results of gene replacement. This result is in sharp contrast to the results of using pJV53 in Example 1.

[0329] The transformant strain containing Donor XXVIII genomic integration verified by PCR was named S11.

[0330] 7.2 Scarless gene editing

[0331] In order to achieve scarless genome editing, after the genome integration of donor XXVIII, the gentamicin resistance cassette needs to be removed by cleavage with the I-SceI homing endonuclease. Recombination between a sequence (SEQ ID NO: 82) encoded by the first donor Donor XXVIII and a sequence in the genome downstream of the downstream homology arm binding region and the corresponding homologous sequence in the genome can guide DNA repair to achieve scarless deletion of the gentamicin resistance cassette without the need to provide a second donor ( Fig.18 ). Therefore, it is necessary to introduce a second plasmid encoding the I-SceI homing endonuclease, i.e. pBotaET39, into the S11 strain.

[0332] S11 (uninduced) was transformed with pBotaET39. The transformant culture was plated on a solid medium containing 50 mg / L kanamycin sulfate and 50 mg / L apramycin sulfate and cultured in a 30°C incubator for 5-8 days.

[0333] Six transformant colonies were randomly selected and tested by PCR using primers P16 and P17. When the template used in the PCR reaction was the same as the genotype of the parent strain S11, a 1.7kb product was produced; when the transformant genome contained the expected gene editing (i.e., the deletion of the gentamicin resistance cassette), a 590bp product was produced ( Fig.18 ). The electrophoresis results of PCR products are as follows Fig. 20 As shown, all 6 transformants were the expected gene editing products. That is, in this experiment, the deletion efficiency of the gentamicin resistance cassette was 100% (6 / 6).

[0334] To confirm that the genome editing that occurred was scarless, the PCR products of two PCR-verified pBotaET39 transformants were randomly selected and Sanger sequenced using primers P16 and P17. Comparison of the sequencing results with the expected editing product sequence confirmed that there was no gentamicin resistance cassette or I-SceI cleavage site in the genome of the selected transformants, and that the editing product sequence was consistent with the expected product sequence ( Fig.21 ).

[0335] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

[0336] Table 7 Sequence information

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

Claims

1. A recombinant engineering system suitable for mycobacteria, comprising an expression system of a gene sequence encoding a RecE protein or its homologue and / or a RecT protein or its homologue, wherein the RecE protein or its homologue and / or the RecT protein or its homologue is derived from a mycobacteriophage, a prophage or a mycobacterium microorganism.

2. The recombineering system according to claim 1, wherein: The RecE protein or its homologue and the RecT protein or its homologue are from the same or different bacteriophages, prophages or bacteria.

3. The recombineering system according to claim 1 or 2, wherein: The RecE protein or its homologue is selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 12, 14, 16, 18, 20, 22, 24 or any variant thereof; The RecT protein or its homologue is selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25 or any variant thereof; Preferably, the RecE protein or a homologue thereof is selected from an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 18, 20, 22, and 24; Preferably, the RecT protein or a homologue thereof is selected from an amino acid sequence having a sequence identity of 64% or more to the amino acid sequence shown in any one of SEQ ID NOs: 19, 21, 23, and 25.

4. The recombineering system according to any one of claims 1 to 3, wherein: The RecE protein and / or RecT protein is selected from any one of the following combinations: (1) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 18, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 19; (2) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 20, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 21; (3) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 22, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 23; (4) a RecE protein having an amino acid sequence as shown in SEQ ID NO: 24, and a RecT protein having an amino acid sequence as shown in SEQ ID NO: 25; (5) A RecT protein having the amino acid sequence shown in SEQ ID NO:

19.

5. The recombineering system according to any one of claims 1 to 4, wherein: The recombineering system is encoded by mobile genetic elements or directly integrated into the host gene.

6. The recombineering system according to any one of claims 1 to 5, wherein: The gene sequence encoding the RecE protein or its homologue and / or the RecT protein or its homologue further comprises a promoter sequence upstream, and the promoter is an inducible promoter or a constitutive promoter; Preferably, the inducible promoter is a tetracycline-inducible promoter, a nitrile-inducible promoter, a thiostrepton-inducible promoter or an acetamide-inducible promoter; Preferably, the constitutive promoter is hsp60 promoter, pms promoter, trp promoter, Psmyc promoter or Pimyc promoter; Preferably, the plasmid comprises a nucleotide sequence selected from any one of SEQ ID NOs: 40-59; Preferably, the plasmid comprises a nucleotide sequence selected from any one of SEQ ID NOs: 49-59.

7. The recombineering system according to claim 1, wherein: The recombineering system further comprises a first recombinant donor, which comprises an upstream homology arm and a downstream homology arm from the 5' end to the 3' end, respectively; Optionally, a mutation sequence is further included between the upstream homology arm and the downstream homology arm, wherein the mutation sequence is used to replace the gene editing target sequence of mycobacterium; Preferably, the first recombination donor is double-stranded DNA or single-stranded DNA; Preferably, the first recombinant donor further comprises at least one cleavage site of a DNA cleavage system, which is located between the upstream homology arm and the downstream homology arm; Preferably, the first recombinant donor further comprises a sequence homologous to the upstream homology arm or the downstream homology arm, which is located between the upstream homology arm and the downstream homology arm; more preferably, the first recombinant donor further comprises at least one cleavage site of a DNA cleavage system, which is located between the upstream homology arm and the sequence homologous to the upstream homology arm, or between the downstream homology arm and the sequence homologous to the downstream homology arm; Preferably, the first recombinant donor also comprises a sequence homologous to a fragment located upstream of the upstream homologous arm binding region in the target molecule, or a sequence homologous to a fragment located downstream of the downstream homologous arm binding region in the target molecule, which is located between the upstream homologous arm and the downstream homologous arm; more preferably, the first recombinant donor also comprises at least one cleavage site of a DNA cleavage system, which is located between the upstream homologous arm and the sequence homologous to a fragment located upstream of the upstream homologous arm binding region in the target molecule, or between the downstream homologous arm and the sequence homologous to a fragment located downstream of the downstream homologous arm binding region in the target molecule.

8. A tool kit for scarless genome editing, comprising the recombineering system of any one of claims 1 to 7, and optionally a DNA cleavage system; Preferably, the DNA cleavage system is selected from a homing endonuclease, a CRISPR-Cas system, a transcription activator-like effector nuclease (TALENs) or a zinc finger nuclease (ZFNs); Preferably, the homing endonuclease is I-SceI; Preferably, the kit further comprises a second recombination donor for directing DNA repair; Preferably, the second recombinant donor comprises an upstream homology arm and a downstream homology arm from the 5' end to the 3' end, respectively; Optionally, the second recombinant donor further comprises a mutant sequence located between the upstream homology arm and the downstream homology arm.

9. A genetically engineered bacterium comprising the recombineering system according to any one of claims 1 to 7 or the tool kit according to claim 8.

10. A method for scarless genome editing, comprising the following steps: Providing a host cell, the host cell comprising the recombineering system of any one of claims 1 to 7 or the tool kit of claim 8; transforming the first recombinant donor into a host cell; Using a recombineering system comprising RecE protein or its homolog and / or RecT protein or its homolog to mediate the recombination of the first recombination donor and the gene editing target molecule; Optionally, the method further comprises the steps of: Utilize DNA cleavage system for targeted DNA cleavage; DNA repair is directed using either the primary recombinant donor or the secondary recombinant donor.

11. The method according to claim 10, comprising the steps of: Providing a host cell, the host cell comprising the recombineering system of any one of claims 1 to 7 or the tool kit of claim 8; Transforming a first recombinant donor into a host cell, wherein the first recombinant donor comprises, from the 5' end to the 3' end, an upstream homology arm, optionally a mutation sequence, a downstream homology arm, and at least one cleavage site located between the upstream homology arm and the downstream homology arm, respectively, and the cleavage site is recognized by a DNA cleavage system; Using a recombineering system comprising RecE protein and / or RecT protein to mediate the recombination of the first recombinant donor and the gene editing target molecule; Utilize DNA cleavage system for targeted DNA cleavage; The second recombinant donor is used to guide DNA repair, wherein the second recombinant donor comprises an upstream homology arm and a downstream homology arm from the 5' end to the 3' end, respectively, and optionally, a mutation sequence located between the upstream homology arm and the downstream homology arm.

12. The method according to claim 10, comprising the steps of: Providing a host cell, the host cell comprising the recombineering system of any one of claims 1 to 7 or the tool kit of claim 8; Transforming a first recombinant donor into a host cell, wherein the first recombinant donor comprises, from the 5' end to the 3' end, an upstream homology arm, an optional mutation sequence, a downstream homology arm, and a sequence homologous to the upstream or downstream homology arm located between the upstream homology arm and the downstream homology arm, and at least one cleavage site located between the upstream or downstream homology arm and the sequence homologous to the upstream or downstream homology arm, and the cleavage site is recognized by a DNA cleavage system; Using a recombineering system comprising RecE protein and / or RecT protein to mediate the recombination of the first recombinant donor and the gene editing target molecule; Utilize DNA cleavage system for targeted DNA cleavage; DNA repair is directed by recombination between sequences homologous to the upstream or downstream homology arms in the first recombination donor and the upstream or downstream homology arms.

13. The method according to claim 10, comprising the steps of: Providing a host cell, the host cell comprising the recombineering system of any one of claims 1 to 7 or the tool kit of any one of claim 8; Transforming a first recombinant donor into a host cell, wherein the first recombinant donor comprises, from the 5' end to the 3' end, an upstream homology arm, an optional mutant sequence, a downstream homology arm, and a sequence between the upstream homology arm and the downstream homology arm homologous to a fragment of the target molecule located upstream of the upstream homology arm binding region, and at least one cleavage site between the upstream homology arm and the downstream homology arm and a sequence homologous to a fragment of the target gene located upstream of the upstream homology arm binding region, or a sequence between the upstream homology arm and the downstream homology arm homologous to a fragment of the target molecule located downstream of the downstream homology arm binding region, and at least one cleavage site between the downstream homology arm and the downstream homology arm and a sequence homologous to a fragment of the target gene located downstream of the downstream homology arm binding region, and the cleavage site is recognized by a DNA cleavage system; Using a recombineering system comprising RecE protein and / or RecT protein to mediate the recombination of the first recombinant donor and the gene editing target molecule; Utilize DNA cleavage system for targeted DNA cleavage; DNA repair is guided by recombination between the homologous sequence described in the first recombination donor and the corresponding homologous sequence upstream or downstream of the target molecule.

14. The method according to any one of claims 10 to 13, wherein: The DNA cleavage system is selected from a homing endonuclease, a CRISPR-Cas system, a transcription activator-like effector nuclease (TALENs) or a zinc finger nuclease (ZFNs); Preferably, the homing endonuclease is I-SceI; Preferably, the host cell is a Mycobacterium microorganism; Preferably, the mycobacterium is a fast-growing mycobacterium, including Mycobacterium smegmatis, Mycobacterium fortuitum, Mycobacterium flavescens, and Mycobacterium neogoldenum; Preferably, the mycobacterium is M. neoaurum strain NRRL B-3805.

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