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CN-122012562-A - CRISPR transposon gene integration system and application thereof

CN122012562ACN 122012562 ACN122012562 ACN 122012562ACN-122012562-A

Abstract

The invention provides a CRISPR transposon gene integration system, which comprises a vector pQCasTns containing transposase and Cas protein and a vector pDoner containing functional sequences, wherein pQCasTns comprises an operon and a T7 terminator which are connected in sequence, and pDoner comprises the functional sequences recognized by the transposase and a crRNA sequence expression module of a target genome. The CRISPR transposon system provided by the invention solves a series of problems existing in halomonas genome editing, and can realize multi-target knockout, efficient deletion of long fragments and efficient integration of exogenous genes.

Inventors

  • JIANG XIAORAN
  • CHENG PING

Assignees

  • 中国人民解放军陆军军医大学

Dates

Publication Date
20260512
Application Date
20260107

Claims (9)

  1. 1. A CRISPR transposon gene integration system comprising: A vector pQCasTns containing a transposase and Cas protein, and a vector pDoner containing a functional sequence; The pQCasTns comprises an operon and a T7 terminator, which are sequentially connected, the operon having the structure: J23119-TnsA-TnsB-TnsC-TniQ-Cas8-Cas7-Cas6; Wherein, J23119 is a J23119 promoter, tnsA-TnsB-TnsC is TnsA, tnsB, tnsC transposase complex derived from vibrio cholerae, tniQ and Cas8-Cas7-Cas6 are Cas6, cas7 and Cas8 protein complex; the functional sequence is a gene cargo sequence insertion site and/or a crRNA expression cassette insertion site; When used for integrating long fragment exogenous genes, the gene cargo sequence is an exogenous gene sequence, the long fragment exogenous gene is greater than 10kb, and/or, When used to delete large genomic fragments, the pQCasTns vector is cloned downstream of the gene with an FLP protein driven by the Mmp1 promoter and the functional sequence of pDoner comprises a first FRT sequence, a target gene and a second FRT sequence, which are sequentially connected, preferably in the opposite direction, the nucleotide sequence of the FRT is SEQ ID NO:30, and/or, When used for integrating exogenous genes at one or more targets, the crRNA expression cassette comprises a promoter and exogenous gene targeting sequences separated by a spacer sequence, wherein the spacer sequence is SEQ ID NO. 38.
  2. 2. The system of claim 1, wherein the operon comprises a gene having a nucleotide sequence of SEQ ID NO. 1-SEQ ID NO. 14.
  3. 3. The system according to claim 1 or 2, wherein the nucleotide sequence of the gene encoding FLP protein is SEQ ID No. 27.
  4. 4. A system according to any one of claims 1 to 3, wherein the gene cargo sequence has upstream recognition site with nucleotide sequence SEQ ID No. 23 and downstream recognition site with nucleotide sequence SEQ ID No. 24 upstream.
  5. 5. The system of any one of claims 1-4, wherein the spacer sequence is SEQ ID NO38.
  6. 6. The system of any one of claims 1-5, wherein the crRNA expression cassette has a nucleotide sequence as set forth in SEQ ID NO. 22.
  7. 7. The system of any one of claims 1-6, wherein the genetic cargo in pDoner is replaced with the target gene sequence by the Gibson assembly technique.
  8. 8. The system of any one of claims 1-7, wherein the pDoner is to replace crRNA sequences with goldengate technology to express crrnas corresponding to different targets.
  9. 9. The use of the system according to any one of claims 1 to 8 in gene editing of halomonas, preferably one or more selected from the group consisting of large genomic fragment deletion, multi-target genomic gene knockout, multi-target exogenous gene integration and long-fragment exogenous gene integration, preferably the large genomic fragment deletion is not less than 119kb, preferably the multi-target exogenous gene integration is simultaneous exogenous gene integration of 2, 3, 4, 5 or 6 targets, preferably the gene length of exogenous long-fragment gene integration is not less than 12kb, preferably the halomonas is a strain with accession number CGMCC No. 4353.

Description

CRISPR transposon gene integration system and application thereof Technical Field The invention relates to the technical field of gene editing. In particular to a CRISPR transposon system derived from vibrio cholerae, and the expression of enzyme in the system is optimized through promoter engineering, thereby realizing the technology of efficiently integrating exogenous genes. Background The halomonas is a halophilic non-model gram-negative bacterium, and because of the special growth environment, namely the optimal growth salt concentration is 40-60g/L and the optimal pH is 8.5-9.0, a plurality of miscellaneous bacteria can not grow, so that open and non-sterile continuous fermentation can be realized, and the cost of industrial production is greatly reduced. Halomonas as an excellent industrial chassis bacteria, there is a need to obtain a range of phenotypes that favor the production of specific products by genome reprogramming. At present, tools for genome knockout or integration in halomonas are limited and have various disadvantages, including 1) a homologous recombination system mediated by suicide plasmids, which has low integration efficiency, 2) CRISPR-Cas9 mediated gene editing, which cannot knockout long fragments and cannot perform multi-target integration and knockout due to the dependence on homologous arms, 3) a base editing system, which can realize multi-target knockout, but is easy to carry out back mutation and cannot integrate exogenous genes, 4) a CRISPRi system, which can realize targeted inactivation of genes by suppressing DNA transcription process through gRNA, but cannot realize genome integration and deletion, and 5) a sRNA suppression system, which can realize reduction of protein expression by suppressing translation process of mRNA through sRNA, and also cannot realize genome integration and deletion. Limitations of the gene editing tools make halomonas difficult and time consuming to engineer. The construction of a highly efficient and versatile gene editing system in halomonas is a major problem to be solved by the present invention. Disclosure of Invention The invention aims to overcome a series of problems existing in the prior art when the halomonas genome is edited, including but not limited to incapability of performing simultaneous multi-target knockout, low efficiency of deleting long fragments, low efficiency of integrating exogenous genes and the like, and provides a technical method capable of efficiently introducing exogenous genes into halomonas. The invention provides a CRISPR transposon genome integration system (CRISPR-Tn system) which can realize efficient genome long fragment integration in halomonas, integrate exogenous genes at more than 3 targets, and realize deletion of genome fragments exceeding 119kb by coupling site-specific recombinase system FLP-FRT. The present invention first provides a CRISPR transposon gene integration system comprising: A vector pQCasTns containing a transposase and Cas protein, and a vector pDoner containing a functional sequence; The pQCasTns comprises an operon and a T7 terminator, which are sequentially connected, the operon having the structure: J23119-TnsA-TnsB-TnsC-TniQ-Cas8-Cas7-Cas6; Wherein, J23119 is a J23119 promoter, tnsA-TnsB-TnsC is TnsA, tnsB, tnsC transposase complex derived from vibrio cholerae, tniQ, and Cas8-Cas7-Cas6 are Cas6, cas7 and Cas8 protein complexes; said pDoner comprises a functional sequence; the functional sequence is a gene cargo sequence insertion site and/or a crRNA expression cassette insertion site; when used for integrating long fragment exogenous genes with length of more than 10kb, the gene cargo sequence is exogenous gene sequence, the long fragment exogenous gene is not less than 12kb, and/or, When used to knock out a genomic target gene, the pQCasTns vector is cloned downstream of the gene with an FLP protein that is driven by the Mmp1 promoter, and the functional sequence of pDoner comprises a first FRT sequence, the target gene and a second FRT sequence that are sequentially linked, preferably in the opposite direction, the nucleotide sequence of the FRT is SEQ ID NO:30, and/or, When used for integrating exogenous genes at one or more targets, the crRNA expression cassette comprises a promoter and exogenous gene sequences separated by a spacer sequence, which is SEQ ID NO. 38. In one embodiment according to the invention pDoner further optionally comprises a resistance gene sequence, an OriT binding transfer element, an ori replicon, a pRO1600 oriV replicon, a pRO1600 rep replication protein encoding gene, preferably said resistance gene sequence is a spectinomycin resistance gene, or/and a kanamycin resistance gene, or/and an erythromycin resistance gene; In one embodiment according to the invention, the operon comprises a gene having the nucleotide sequence of SEQ ID NO. 1-SEQ ID NO. 14. In one embodiment according to the invention, the nucleotide sequence of the gene encoding th