Traceless double-target genome editing system

A genome editing, dual-target technology, applied in the field of genome editing, can solve the problems of resistance gene removal, difficulty, and insufficiency

Active Publication Date: 2019-05-31
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this efficiency is still difficult to screen for positive recombinants. The first commonly used method is to use the resistance gene as a screening marker. The disadvantage of this method is that the subsequent resistance gene needs to be eliminated; the second method is ...

Method used

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  • Traceless double-target genome editing system
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  • Traceless double-target genome editing system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0044] Implementation case 1. Simultaneously gal K (PAM dependent) and lac Z gene (PAM-independent) point mutation

[0045] Escherichia coli E. coli W3110

[0046] Table 1 Primers and related sequences required for double point mutation

[0047]

[0048] (1) Transformation of pCSK plasmid: Use the primer sequences in Table 1 to carry out pCSK plasmid gal K and lac Z Addition of target sequence gRNA: the specific method is as follows:

[0049] gal The K sgRNA was added behind the tetracycline promoter using the USER-LIC cloning method. The specific step is to combine two new target gal The K sgRNA complementary single strand was treated in a water bath at 95°C for 5 min and then annealed overnight, then mixed with the pCSK product amplified by the pCSK primers, treated with USER enzyme and then transferred to DH5a for identification to complete the pCSK plasmid backbone and gal K sgRNA binding.

[0050] lac The position of the Z crRNA target is behind the...

Embodiment example 2

[0055] Implementation case two, at the same time gal K gene and ATP-dependent RNA helicase dbp A gene replacement

[0056] Adopt bacterial strain DH5a, adopt table 2 primer and sequence:

[0057] Table 2 Simultaneous gene replacement and gene insertion primers and sequences

[0058]

[0059] The research method is the same as the implementation case 1, of which about 700bp gal K gene was replaced by 1247bp fragment, 1000bp dbpA gene is 560bp lacZ ' DNA fragment substitution. because lac The insertion of Z' can realize α-complementation in the DH5a genome, so that the mutants can form blue colonies on the LB plate containing IPTG and XGal. for gal The identification after the K gene replacement still uses the MacConkey red and white spot method for preliminary screening. In addition, the homologous recombination sequence used this time is a double-stranded DNA PCR product with 40bp homology arms on both sides. gal The K. del700bp.HP primer produces a fragme...

Embodiment example 3

[0062] Implementation case 3. Simultaneous lactosinase gal K gene replacement and β-galactosidase lac Z gene point mutation

[0063] Strains used: E. coli W3110, using primers and DNA sequences from Table 1 lac Part Z and Table 2 of gal K part composition.

[0064] The method adopted is the same as that of the implementation cases 1 and 2.

[0065] experiment result shows( Figure 8 ): gal The mutation efficiency of K is 61.4%, lac The mutation efficiency of Z was 95.1%.

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Abstract

The invention discloses a traceless double-target genome editing system based on CRISPR/Cas9 and lambda-Red. The system is characterized in that 1, pKS9 has the molecular weight of 11,681 kb, is a thermo-sensitive plasmid, and contains cas9 and recX genes induced by tetracycline, lambda-Red integrity alpha-beta-gamma recombinase genes induced by arabinose, and resistance genes ampr; 2, pCSK has the molecular weight of 2,914 kb, and contains two sgRNA insertion sites for specific recognition of genomes, two spacer region gRNA sequences which are induced by rhamnose and used for plasmid elimination and resistance genes kanr; 3, when a homologous arm is 27 bp, the two-point mutation efficiency is both at 92% or above; when the homologous arm is 40 bp, the 1kb replacement efficiency of the twogenes is 60% or above, and when large-fragment gene replacement and point mutation are carried out simultaneously, the respective editing efficiency is not influenced.

Description

technical field [0001] The invention belongs to the technical field of genome editing, and in particular relates to an efficient and traceless genome editing method based on the combination of bacterial CRISPR / Cas9 type II immune system and λ-Red DNA recombination system. Background technique [0002] CRISPR (Clustered regulatory interspaced short palindromic repeats), which can be literally translated as clustered, regularly spaced short palindromic repeats, is a highly efficient gene cutter widely present in the genomes of bacteria and archaea, used to resist Invasion of foreign genetic material such as phages. Structurally, the system consists of a leader region (Leader), multiple highly conserved short repeat sequence regions (Repeat) and multiple spacers (Spacer) existing in the repeat sequence interval. The leader region is rich in AT and has a length of 300-500 bp, which is generally located upstream of CRISPR and is considered to be the promoter sequence of CRISPR. ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/90
Inventor 郑继平郭桂英曾纪锋杨诺周楚新李迁
Owner HAINAN UNIVERSITY
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