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III-A type CRISPR-Cas system inhibitor AcrIIIA2 and application thereof

A III-A, 1.III-A technology, applied in the field of RNA editing, can solve problems such as unknown consequences

Pending Publication Date: 2021-06-18
西部(重庆)科学城种质创制大科学中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the continuous activation of the Cas protein after completing the editing of the target gene, it can lead to non-specific cleavage at the entire gene level, resulting in unknown consequences

Method used

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  • III-A type CRISPR-Cas system inhibitor AcrIIIA2 and application thereof
  • III-A type CRISPR-Cas system inhibitor AcrIIIA2 and application thereof
  • III-A type CRISPR-Cas system inhibitor AcrIIIA2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Identification and cloning of AcrIIIA1 and AcrIIIA2

[0023] (1) Biological information screening and analysis of candidate AcrIIIAs genes

[0024] According to the presence of RNA sequences that can be targeted and recognized by type III-A CRISPR-Cas in the bacterial transcriptome, it is suggested that the bacteria's own genome may contain inhibitors that prevent the RNA editing activity of type III-A CRISPR-Cas systems. According to this principle, in the Staphylococcus argenteus 3688STDY6125118 bacterial gene sequence (https: / / www.ncbi.nlm.nih.gov / , accession number: NZ_FQLY01000002.1) containing the III-A type CRISPR-Cas system, through Self-Targeting Space Search Platform (SSTS) analysis to obtain sequence sites that can be targeted and recognized by Type III-A CRISPR-Cas ( figure 1 Middle A), implying that there may be an inhibitor of RNA-cleaving activity of type III-A CRISPR-Cas in S. argenteus 3688STDY6125118. Because the Acr gene usually clusters with the Ac...

Embodiment 2

[0037] AcrIIIA1 and AcrIIIA2 inhibit RNA-cleaving activity of type III-A CRISPR-Cas in bacteria

[0038] To verify whether AcrIIIA1 and AcrIIIA2 inhibit the activity of type III-A CRISPR-Cas in bacteria. First, the pCRISPR MS2 recombinant plasmid was constructed to obtain the crRNA expression vector used to recognize the rep RNA of MS2 RNA phage. The pre-crRNA sequence is synthesized by GeneUniversal, such as figure 2 As shown in A, the pre-crRNA was linked to the pBluescript II sk(+)1 vector by T4 DNA ligase to obtain the recombinant plasmid pCRISPR MS2. Secondly, pET28a-AcrIIIA1, pET28a-AcrIIIA2 or pET28a, T7-pCas / Csm plasmid and pCRISPR MS2 plasmid were transformed into competent cells to obtain E. coli c-3000 / T7-pCas / Csm / pCRISPR MS2 / pET28a, E. The coli c-3000 / T7-pCas / Csm / pCRISPR MS2 / pET28a-AcrIIIA1 and E.coli c-3000 / T7-pCas / Csm / pCRISPR MS2 / pET28a-AcrIIIA2 strains were used for the plaque formation experiment of MS2 RNA phage invasion ( figure 2 Middle A). The obtain...

Embodiment 3

[0041] AcrIIIA1 and AcrIIIA2 inhibit the editing activity of type III-A CRISPR-Cas RNA in mammalian cells

[0042] In order to verify whether AcrIIIA1 and AcrIIIA2 inhibit type III-A CRISPR-CasRNA editing activity in HEK293T cells, the effect of AcrIIIA1 and AcrIIIA2 on type III-A CRISPR-Cas cutting influenza virus (Influenza A virus, IAV) was detected in HEK293T cells . Using Lipofectamine TM CRISPRMAX TM Transfection Reagent (Thermo Fisher Scientific) transfected HEK293T cells with type III-A CRISPR-Cas complex targeting IAV and AcrIIIA1 or AcrIIIA2 protein, then infected IAV virus (MOI=0.01 and 0.5, multiplicity of infection), and detected IAV virus RNA and virus titers ( image 3 Middle A). The process is as follows:

[0043] 1) Ni column affinity chromatography purification of AcrIIIA1, AcrIIIA2 and III-A type CRISPR-Cas proteins:

[0044] The constructed pET28a-His-AcrIIIA1, pET28a-His-AcrIIIA1, and T7-pCas / Csm / His-Csm2 plasmids were transformed into E.coli BL21 c...

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Abstract

The invention belongs to the technical field of RNA editing, and particularly relates to a III-A type CRISPR-Cas system inhibitor AcrIIIA2 and application thereof. According to the invention, the III-A type CRISPR-Cas system inhibitor AcrIIIA2 for inhibiting the editing activity of a III-A type CRISPR-Cas gene is screened from a Staphyloccus argenteus 3688STDY6125118 bacterial gene sequence, and in bacteria andmammalian cells, the AcrIIIA2 controls "closing" of the III-A type CRISPR-Cas gene editing ability. In addition, the invention further provides a homologous protein of the AcrIIIA2. The homologous protein has the same inhibition effect, and the variety of III-A type CRISPR-Cas system inhibitors is enriched. The AcrIIIA2 inhibitor and the homologous protein thereof can control the III-A type CRISPR-Cas RNA editing efficiency in time or space, and the safety and practicability of the III-A type CRISPR-Cas editing technology in the fields of biological treatment, biotechnology, agriculture and the like are improved.

Description

technical field [0001] The invention belongs to the technical field of RNA editing, in particular to a type III-A CRISPR-Cas system inhibitor AcrIIIA2 and its application. Background technique [0002] One of the most exciting discoveries in microbiology in the past decade is that, similar to eukaryotes, bacteria also have an acquired immune system, breaking the long-standing conclusion that "acquired immunity is unique to eukaryotes". In the process of foreign invasion, bacteria have evolved a new and unique immune defense system, namely the CRISPR-Cas system (clustered regularly interspaced short palindromic repeats and related Cas proteins). The CRISPR-Cas system protects bacteria and archaea from the invasion of foreign phages, viruses and plasmids by capturing and integrating exogenous nucleic acid fragments, and under the joint action of Cas protein and CRISPR RNAs (crRNA). CRISPR-Cas systems have been developed for gene editing, with applications in bioscience, medic...

Claims

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Application Information

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IPC IPC(8): C07K14/31C12N9/22C12N15/113
CPCC07K14/31C12N9/22Y02A50/30
Inventor 林平蒋建新吴敏
Owner 西部(重庆)科学城种质创制大科学中心