PCr-NHEJ (non-homologous end joining) carrier as well as construction method of pCr-NHEJ carrier and application of pCr-NHEJ carrier in site-specific knockout of bacterial genes

A construction method and a technology for targeted knockout, applied in the field of genetic engineering, can solve the problems of cumbersome operation, difficult operation, and insufficient gene knockout efficiency, and achieve the effects of low operation difficulty, reduced operation difficulty, and high gene knockout efficiency.

Inactive Publication Date: 2015-06-03

GUANGDONG MEDICAL UNIV

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AI-Extracted Technical Summary

Problems solved by technology

Therefore, the CRISPR-Cas9 system of the prior art has the disadvantages of cumb...

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Abstract

The invention belongs to the technical field of gene engineering, in particular to a pCr-NHEJ (non-homologous end joining) carrier as well as a construction method of the pCr-NHEJ carrier and an application of the pCr-NHEJ carrier in site-specific knockout of bacterial genes. The sequence of the pCr-NHEJ carrier is shown as the SEQ ID NO.1. The application of the pCr-NHEJ carrier in site-specific knockout of the bacterial genes is provided. The technical principle is as follows: NHEJ and CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated protein 9) are jointly applied, so that after DNA (deoxyribonucleic acid) is broken when a CRISPR-Cas9 system cuts bacterial genomic DNA, the NHEJ system can be connected with a broken DNA end automatically, bacteria are survived, double-strand DNA having homology with a target sequence is not required to be introduced artificially to repair the broken DNA end, and the operation steps of the CRISPR-Cas9 technique are simplified.

Application Domain

BacteriaVector-based foreign material introduction

Technology Topic

CRISPR-Associated ProteinsUnclassified Bacteria +9

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Examples

Experimental program(5)
Effect test(1)

Example Embodiment

[0060] Example 1.

[0061] A pCr-NHEJ vector, the sequence of the pCr-NHEJ vector has the sequence shown in SEQ ID NO:1. The pCr-NHEJ vector of this embodiment can be used for bacterial gene knockout, and has the advantage of high gene knockout efficiency.

Example Embodiment

[0062] Example 2.

[0063] The application of a pCr-NHEJ vector of Example 1 in site-specific knockout of bacterial genes.

Example Embodiment

[0064] Example 3.

[0065] The construction method of a pCr-NHEJ vector of Example 1, includes the following steps:

[0066] (1) Use broad host plasmid of gram-negative bacteria P BBR1MCS-2 (GI: 773412) as the basic carrier; cas9

[0067] (2) The following three protein genes were amplified using the upstream primer and downstream primer with the prokaryotic promoter PLtetO-1 for mRNA transcription: cas9 (GI:674296984), ku (GI:444893469) and ligD (GI:444893469), respectively cas9 Amplification products, ku Amplification products and ligD Amplification product; among them, cas9 , ku with ligD The open reading frames of the three protein genes are all located downstream of independent PLtetO-1, so that cas9 , ku with ligD The expression of the three protein genes is regulated by the promoter; cas9 Amplification products, ku Amplification products and ligD There is a 15nt base overlap between the amplified products;

[0068] (3) cas9 The 3’ end of the amplified product and ku The 5'end of the amplified product, ku The 3’ end of the amplified product and ligD Between the 3'ends of the amplified products, there is a 15nt base overlap, which is connected by in-fusion PCR cas9 Amplification products, ku Amplification products and ligD Amplify the product to obtain the first linker;

[0069] (4) Use restriction endonuclease KpnI and HindIII to double digest the basic vector P BBR1MCS-2, the digestion product of the basic vector is obtained; the digestion product is subjected to gel electrophoresis, the gel is cut, and the digestion product is purified by a DNA recovery kit; then the purified digestion product is connected with the first connection by in-fusion PCR To obtain the vector pNHEJ;

[0070] (5) Digest the vector pNHEJ with NotI and SpeI to obtain the digested product of the vector pNHEJ;

[0071] (6) Simultaneously introduce two adjacent inverted BsaI restriction sites and two adjacent inverted BsmAI restriction sites into the vector pNHEJ for transcription to form sgRNA that can simultaneously bind to two positions of the target gene; Among them, the DNA fragment with two adjacent inverted BsaI restriction sites is DBsaI, and the DNA fragment with two adjacent inverted BsmAI restriction sites is DBsmAI;

[0072] (7) Link DBsaI and DBsmAI by in-fusion PCR to obtain the second ligation product;

[0073] (8) Connect the second ligation product and the digested product of the vector pNHEJ by in-fusion PCR to obtain the CRISPR-Cas9 system for knocking out the target gene, that is, the pCr-NHEJ vector.

[0074] Wherein, in step (2), the primer PPLtetO-1-cas9U with the sequence shown in SEQ ID NO: 2 and the primer PCas9D with the sequence shown in SEQ ID NO: 3 are used for amplification cas9 Protein gene, get cas9 Amplification product

[0075] Use primer PPLtetO-1-KuU with the sequence shown in SEQ ID NO: 4 and primer PkuD with the sequence shown in SEQ ID NO: 5 for amplification ku Protein gene, get ku Amplification product

[0076] Amplify with primer PLtetO-1-ligDU having the sequence shown in SEQ ID NO: 6 and primer PligDD having the sequence shown in SEQ ID NO: 7 ligD Protein gene, get ligD Amplification product.

[0077] Among them, in step (6), the DNA fragment DBsaI with two adjacent inverted BsaI restriction sites is constructed as follows:

[0078] First, directly synthesize the DNA sequence BsaIU with the sequence shown in SEQ ID NO: 8 and the DNA sequence BsaID with the sequence shown in SEQ ID NO: 9, wherein the 3'end of BsaIU and the 5'end of BsaID have 15 nt overlapping sequences; Among them, BsaID contains tracrRNA sequence;

[0079] Connect BsaIU and BsaID by in-fusion PCR; then use the primer PBsaIU with the sequence shown in SEQ ID NO: 10 and the primer PBsaID with the sequence shown in SEQ ID NO: 11, PCR amplification, that is, the adjacent The two inverted BsaI restriction sites of the DNA fragment DBsaI.

[0080] Among them, in step (6), the construction of the DNA fragment DBsmAI with two adjacent inverted BsmAI restriction sites is as follows:

[0081] First, directly synthesize the DNA sequence BsmAIU with the sequence shown in SEQ ID NO: 12 and the DNA sequence BsmAID with the sequence shown in SEQ ID NO: 13; wherein, BsmAID contains the tracrRNA sequence;

[0082] The DNA sequence BsmAIU and the DNA sequence BsmAID are connected by in-fusion PCR; then the primer PBsmAIU with the sequence shown in SEQ ID NO: 14 and the primer PBsmAID with the sequence shown in SEQ ID NO: 15 are used for PCR amplification, namely A DNA fragment DBsmAI with two adjacent inverted BsmAI restriction sites was obtained.

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Classification and recommendation of technical efficacy words

High gene knockout efficiency
Easy to operate

gRNA subjected to wild type T cell TCR alpha chain knockout and method

InactiveCN107236741AHigh gene knockout efficiencyEasy to prepareImmunoglobulin superfamilyStable introduction of DNAMolecular biologyT cell immunity

Owner:THE FIFTH AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV

PCr-NHEJ (non-homologous end joining) carrier as well as construction method of pCr-NHEJ carrier and application of pCr-NHEJ carrier in site-specific knockout of bacterial genes

AI-Extracted Technical Summary

Problems solved by technology

Abstract

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Examples

Example Embodiment

Example Embodiment

Example Embodiment

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Description & Claims & Application Information

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