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CN-121975826-A - Anti-leakage Cre recombination system and editing method thereof

CN121975826ACN 121975826 ACN121975826 ACN 121975826ACN-121975826-A

Abstract

The invention belongs to the technical fields of synthetic biology and genetic engineering, and particularly discloses an anti-leakage Cre recombinase expression frame and application thereof in pichia pastoris gene editing. The expression frame consists of lox 71-screening mark-sh ble-Cre coding sequence (SEQ ID NO: 1) -TetO operating sequence-lox 66 optimized by pichia pastoris codon and introduced with rare codon of escherichia coli, and completely eliminates PaqCI, bsmBI, bsaI, aarI and other IIS type restriction enzyme sites, and clones the expression frame to pPICZ skeleton and introduces the pPICZ skeleton into the escherichia coli host integrating tetR, so that Cre leakage expression can be completely inhibited in a large-scale amplification stage, and recombination toxicity and plasmid mutation are avoided. The construction of an upstream homology arm-expression frame-downstream homology arm edit frame can be obtained by virtue of Golden Gate one-step assembly, the construction is completed within 3 days, pichia pastoris is electrically transformed after linearization, the first integration efficiency is more than 70%, cre self-deletion is induced by methanol, the resistance mark removal rate is 100%, and only one lox71/66 heterozygous site is reserved. The invention solves the problems of easy mutation of the traditional Cre/loxP system in escherichia coli and low efficiency in yeast, and provides a high-efficiency and safe universal gene editing tool for industrial microorganism metabolic engineering.

Inventors

  • Zhang gun
  • XIE HAISONG
  • DING XU
  • SUN JINWANG
  • SONG QIANQIAN

Assignees

  • 杭州中美华东制药有限公司

Dates

Publication Date
20260505
Application Date
20260115

Claims (12)

  1. 1. An anti-leakage Cre recombinase expression cassette, characterized in that the expression cassette comprises, in order along the 5 '. Fwdarw.3': (i) lox71 site; (ii) Screening the marker genes; (iii) A Cre recombinase coding sequence pCre optimized by pichia pastoris codons and introduced with rare codons of escherichia coli; (iv) TetO manipulation sequences; ( ) lox66 site; Wherein the pCre sequence is shown as SEQ ID NO. 1, and the whole expression frame does not contain a PaqCI, bsmBI, bsaI, aarI recognition site.
  2. 2. The expression cassette of claim 1, wherein the selectable marker gene is sh ble, conferring Zeocin resistance.
  3. 3. The expression cassette of claim 1 or 2, wherein the TetO sequence is 2 x TetO, located between the AOX1 promoter downstream and the pCre start codon.
  4. 4. A vector comprising the anti-leakage Cre recombinase expression cassette of any one of claims 1-3 wherein the vector backbone has eliminated the BsmBI/AarI sites.
  5. 5. The vector of claim 4, wherein the vector is pPICZ-tetO-pCre, and the complete sequence is shown in SEQ ID NO. 3.
  6. 6. A host bacterium, wherein the host bacterium chromosome is integrated with a tetR gene and is transformed with the vector of claim 4 or 5, and the host bacterium is used for strictly inhibiting Cre leakage expression in the stage of escherichia coli.
  7. 7. The host bacterium according to claim 6, wherein the host bacterium is TOP10F' -tetR.
  8. 8. A gene editing frame, characterized in that the editing frame is obtained by one-step assembly of Golden Gate from the following modules: (i) Upstream homology arm UP of target gene; (ii) The leakage-resistant Cre recombinase expression cassette of any one of claims 1-3; (iii) Downstream homology arm DW of target gene; The UP and the DW are seamlessly inserted into the expression frame through PaqCI/BsaI sites to form a UP-lox71-sh ble-tetO-pCre-lox66-DW structure.
  9. 9. The gene edit box of claim 8, further linearized by SpeI for use in pichia pastoris electrotransformation.
  10. 10. A method for knocking out a target gene of pichia pastoris, comprising: (i) Electrically converting the linearization edit box of claim 8 or 9 to pichia pastoris X33; (ii) Screening for first integration events using Zeocin resistance; (iii) The tetO-pCre is expressed through methanol induction, lox71/66 recombination is driven, and the screening mark is automatically removed; (iv) The complete deletion of the open reading frame of the target gene and the retention of only one lox71/66 heterozygous site were verified by PCR and sequencing.
  11. 11. The method of claim 10, wherein the first integration efficiency is greater than or equal to 70% and the mark removal efficiency is 100%.
  12. 12. Use of the leakage-resistant Cre recombinase expression cassette of any one of claims 1-3, the vector of claim 4 or 5, the host bacterium of claim 6 or 7, or the gene editing cassette of claim 8 or 9 in pichia pastoris gene editing.

Description

Anti-leakage Cre recombination system and editing method thereof Technical Field The invention belongs to the fields of synthetic biology and genetic engineering, and particularly relates to an anti-leakage Cre recombinase expression frame, a matched vector and host bacteria, and a method for realizing rapid and gene editing in pichia pastoris by using the expression frame. Background The Cre/loxP system is widely used for gene knockout, marker recovery and metabolic pathway regulation due to its simple composition and no need of cofactors. However, leakage expression of Cre recombinase in E.coli can lead to plasmid mutation, cytotoxicity and subsequent edit box construction failure, and in Pichia pastoris, large fragment editing is particularly difficult due to low homologous recombination efficiency and high risk of CRISPR/Cas9 off-target. The prior art still cannot meet three requirements of residue-free, high efficiency and easy operation through strategies such as mutant loxP, double-antibiotic reverse selection or secondary transformation. Therefore, there is a need for a general solution that can completely inhibit Cre leakage during the E.coli phase, can induce expression during the yeast phase, and can accomplish self-deletion of the markers at one time. Disclosure of Invention Aiming at the defects existing in the prior art, the primary aim of the invention is to provide an anti-leakage Cre recombinase expression frame which can keep zero background expression in the amplification stage of escherichia coli and realize high-efficiency recombination after methanol induction in pichia pastoris. The second object of the present invention is to provide a vector, a host bacterium and a gene editing box containing the expression box, so that the assembly of large fragment DNA can be completed within 3 days, and the final product has no foreign sequence residue. The third object of the invention is to provide the Pichia pastoris target gene knockout method based on the elements, the integration efficiency is more than 70%, the mark removal rate is 100%, and a universal tool is provided for industrial microorganism metabolic engineering. The invention realizes the aim through the following specific technical scheme: 1. Expression frame design The expression frame sequentially comprises a lox71 site, a screening marker gene, a Cre recombinase coding sequence pCre (SEQ ID NO: 1) optimized by pichia pastoris codons and introduced with rare codons of escherichia coli, a TetO manipulation sequence (preferably 2×TetO) and a lox66 site along the 5 '. Fwdarw.3' direction, wherein the whole expression frame does not contain PaqCI, bsmBI, bsaI, aarI recognition sites, and the screening gene is preferably sh ble, so that the Zeocin resistance is endowed. 2. Vectors and hosts The above expression cassette was inserted into the BstBI/EcoRI site of pPICZ. Alpha.A, and TetO was introduced downstream of the AOX1 promoter, and the backbone BsmBI/AarI site was eliminated to give pPICZ-tetO-pCre (SEQ ID NO: 3). The TOP10F ' host bacteria with chromosome integrated tetR are selected to form a strict regulation system of ' TetR-2×TetO ', so that ' zero ' leakage expression of Cre recombinase in the escherichia coli stage is realized. 3. Edit box construction The Golden Gate reaction is utilized to assemble the upstream homology arm UP, the expression frame CORE and the downstream homology arm DW to a pUC-IIS (Kan≡R, no IIS site) framework through PaqCI/BsaI sites in one step to obtain pUC-IIS-CORE-UD, and the linear editing frame of 8.0 kb is obtained after SpeI linearization, so that the method can be directly used for yeast transformation. 4. Pichia pastoris transformation and self-deletion After the linear editing frame is electrically converted into X33, screening a first integration event by means of sh ble, then driving lox71/66 to recombine by means of methanol induction and tetO-pCre, automatically removing a resistance mark, and only reserving one lox71/66 heterozygous site to realize knockout. 5. Effect data (1) Passage stability the control plasmid pPICZ alpha A-cre accumulated 8.7 mutations on average in passage 7, while pPICZ-tetO-pCre had zero mutations in passage 7; (2) Integration efficiency 73.4% of the first integration efficiency of the target gene locus (14/19); (3) Marker removal 3 independent clones were all successfully stripped of sh ble and no additional mutations were verified by PCR sequencing. The invention has the beneficial effects that: (i) The Cre zero leakage in the escherichia coli stage thoroughly solves the problems of plasmid mutation and cytotoxicity; (ii) The editing frame assembly is completed within 3 days, so that long fragment PCR is avoided, and the mutation rate is reduced by 1-2 orders of magnitude; (iii) The yeast stage realizes integration-self deletion at one time, does not leave any exogenous sequence, and meets the requirements of biosafety and regulations; (iv) The coexistenc