VI-B type CRISPR/Cas13 gene editing system and application thereof

A VI-B, gene editing technology, applied in the field of gene editing

Active Publication Date: 2021-03-02
ZHUHAI SHU TONG MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In prokaryotes, this system can target and edit single-stranded RNA by recognizing and cutting the protospacer flanking site PFS (Protospacer flanking site) sequence on both sides of the targeted polynucleotide, while in eukaryotes In biology, the targeted editing of RNA by type VI-B CRISPR / Cas system has no PFS sequence

Method used

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  • VI-B type CRISPR/Cas13 gene editing system and application thereof
  • VI-B type CRISPR/Cas13 gene editing system and application thereof
  • VI-B type CRISPR/Cas13 gene editing system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This embodiment analyzes and predicts the genes related to Lt1Cas13b described in the present invention.

[0039] (1) Materials: metagenomic sequencing data.

[0040] (2) Software: fastqc (v0.11.5), fastp (v0.19.8), SOAPnuke (v1.5.2), hista2 (v2.0.4), samtools (v1.9), iTools (v0.23), GeneMark (v3 .38), IDBA-UD(v1.1.3), Bowtie2(v2.3.5.1), CD-HIT(v4.6), hummer(3.1b2), BLAST(v2.3.0+),Ordination(v1.0 ), WilcoxonTest (v1.0), Environmental CorrAnalysis (v1.0)

[0041] (3) Detection method

[0042] The data source of this embodiment is metagenomic sequencing data, and all possible gene sequences in the metagenomic set are obtained by performing quality control, splicing and gene prediction on the metagenomic data.

[0043] For the raw data of the metagenomic group, the specific operation is as follows:

[0044] (a) First use fastqc to charge, check whether the data is original data or cleaned data, if it is original data, then use fastp to clean the original data and remov...

Embodiment 2

[0051] In this example, the obtained contig that may be related to the type 2 CRISPR-Cas system is analyzed, and the predicted protein and related elements related to the CRISPR-Cas system are obtained; that is, the contig obtained in Example 1 is related to CRISPR / Cas13 CRISPR sequence and Cas13 protein prediction.

[0052] (1) Materials: the contig sequence obtained in Example 1 that may be related to the CRISPR-Cas system.

[0053] (2) Software: CRISPR CasFinder (v4.2.19), python (v3.7.4), bedtools (v2.25.0).

[0054] (3) Detection method:

[0055] The specific operation is as follows:

[0056] (a) Use CRISPRCasFinder to analyze and predict the obtained contig that may be related to the type 2 CRISPRCasFinder system, and obtain Cas protein, Spacer, direct repeat and other CRISPR-Cas system related proteins and elements;

[0057] (b) Annotate the subclassification of candidate type VI protein families: collect the HMM files of Cas13a, Cas13b, Cas13c and Cas13d effector pr...

Embodiment 3

[0061] This embodiment is to predict the RNA secondary structure of the guide RNA molecule recognized by the Cas13b gene editing system of the present invention, and the obtained RNA secondary structure is as follows: figure 2 shown.

[0062] (1) Material: repeat sequence;

[0063] (2) Software: NUPACK (http: / / www.nupack.org / partition / new);

[0064] (3) Prediction method: By using the online application NUPACK to simulate the secondary structure of repeat RNA in vitro at 37°C, the figure 2 RNA secondary structures shown.

[0065] Depend on figure 2 It can be seen that the pre-crRNA forms a mature crRNA under the action of Lt1Cas13 nuclease, forming a guide RNA (guide RNA), and the guide RNA includes two stem-loop structures.

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Abstract

The invention relates to the technical field of gene editing, in particular to a VI-B type CRISPR / Cas13 gene editing system and application thereof. The VI-B type CRISPR / Cas13 gene editing system disclosed by the invention comprises an Lt1Cas13b protein and CRISPR RNA; and the Lt1Cas13b protein is RNA endonuclease, and the Lt1Cas13b protein has an amino acid sequence as shown in SEQ ID NO: 1 or anamino acid sequence which is at least 80% the same as the amino acid sequence as shown in the SEQ ID NO: 1. The invention digs out the novel VI-B type CRISPR / Cas13 gene editing system. The system canbe widely applied to scenes needing to identify, combine and edit prokaryote or eukaryote RNA, and provides a new application choice for an RNA editing tool.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to a VI-B type CRISPR / Cas13 gene editing system and its application. Background technique [0002] Gene editing technology makes it possible to modify DNA sequence positioning points, such as zinc finger nucleases (zinc finger nucleases, ZFNs) in the first generation of gene editing tools, and small nucleases similar to transcription activation in the second generation of gene editing tools ( transcription activator-like effector nucleases, TALENs), type II and type V CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat, Clustered Regularly Interspaced Short Palindromic Repeat) / Cas (CRISPR- associated protein) can be used to target the genome, but these gene editing systems can only target the genome and foreign DNA, but cannot target the editing of foreign RNA. [0003] Type VI CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) / Cas (CRISPR-associate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/70C12N15/85C12R1/19
CPCC12N9/22C12N15/70C12N15/85C12N2800/107
Inventor 胡争崔资凤黄昭玥李利芳
Owner ZHUHAI SHU TONG MEDICAL TECH CO LTD
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