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CRISPR-Cas12j enzymes and systems

A direct, C-terminal technology, applied in the field of nucleic acid editing and regularly clustered interspaced short palindromic repeats, which can solve problems such as reducing off-target effects

Active Publication Date: 2021-10-01
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the PAM sequences of Cas9, Cpf1, CasX, and CasY are relatively complex and diverse, while C2c1 recognizes strict 5'-TTN, so its target site is easier to predict than other systems, reducing potential off-target effects

Method used

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  • CRISPR-Cas12j enzymes and systems
  • CRISPR-Cas12j enzymes and systems
  • CRISPR-Cas12j enzymes and systems

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0542] Example 1. The acquisition of Cas12j gene and Cas12j guide RNA

[0543] 1. CRISPR and gene annotation: use Prodigal to annotate the microbial genome and metagenomic data of NCBI and JGI databases to obtain all proteins, and use Piler-CR to annotate CRISPR loci, and the parameters are default parameters.

[0544] 2. Protein filtering: De-redundant annotated proteins through sequence consistency, remove proteins with completely identical sequences, and classify proteins longer than 800 amino acids into macromolecular proteins. Since all the effector proteins of the second type of CRISPR / Cas system discovered so far are more than 900 amino acids in length, in order to reduce the computational complexity, we only consider macromolecular proteins larger than 800 amino acids when mining CRISPR effector proteins.

[0545] 3. Acquisition of CRISPR-related macromolecular proteins: Extend each CRISPR locus upstream and downstream by 10Kb, and identify non-redundant macromolecular...

Embodiment 2

[0550] Example 2.Cas12j gene processing of pre-crRNA

[0551] 1. In vitro expression and purification of Cas12j protein

[0552] The in vitro expression and purification steps of Cas12j protein are as follows:

[0553] 1. Artificially synthesize the DNA sequence encoding the Cas12j protein (SEQ ID NO:82-101) with nuclear localization signal.

[0554] 2. Ligate the double-stranded DNA molecule synthesized in step 1 with the prokaryotic expression vector pET-30a(+) to obtain the recombinant plasmid pET-30a-CRISPR / Cas12j.

[0555] 3. The recombinant plasmid pET-30a-CRISPR / Cas12j was introduced into Escherichia coli EC100 to obtain recombinant bacteria, which were named EC100-CRISPR / Cas12j.

[0556] A single clone of EC100-CRISPR / Cas12j was taken, inoculated into 100 mL LB liquid medium (containing 50 μg / mL ampicillin), and cultured with shaking at 37°C and 200 rpm for 12 hours to obtain a culture liquid.

[0557] 4. Take the culture solution, inoculate it into 50 mL of LB liqu...

Embodiment 3

[0611] PAM domain identification of embodiment 3.Cas12j protein

[0612] 1. Construct the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12j and sequence it. According to the sequencing results, the structure of the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12j is described as follows: the small fragment between the restriction endonuclease Pml I and Kpn I recognition sequences of the vector pACYC-Duet-1 is replaced with the Cas12j gene ( The double-stranded DNA molecule shown in the first position from the 5' end to the last position in the 3' end in the sequence shown in SEQ ID NO:21-40). The recombinant plasmid pACYC-Duet-1+CRISPR / Cas12j expresses Cas12j protein (SEQ ID NO:1-20, 107, 108) and Cas12j guide RNA shown in SEQ ID NO:104.

[0613] 2. The recombinant plasmid pACYC-Duet-1+CRISPR / Cas12j contains an expression cassette, the nucleotide sequence of which is composed of the Cas12j gene and SEQ ID NO: 104 respectively. For example shown in SEQ ID NO:102. In the sequence ...

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Abstract

The invention provides a CRISPR-Cas12j (Clustered Regularly Interspaced Short Palindromic Repeats in particular, the present invention provides a Cas effector protein, a fusion protein such a protein, and a nucleic acid molecule encoding the same. Also provided are complexes and compositions for nucleic acid editing, such as complexes and compositions for gene or genome editing, a Cas effector protein or fusion protein, or a nucleic acid molecule encoding the same. Also provided are methods for nucleic acid editing, such as gene or genome editing, using Cas effector proteins or fusion proteins.

Description

[0001] This application is a divisional application of the invention application with the application number 201980014005.0, the application date is November 15, 2019, and the invention name is "CRISPR-Cas12j enzyme and system". technical field [0002] The invention relates to the field of nucleic acid editing, in particular to the technical field of clustered regularly interspaced short palindromic repeats (CRISPR). In particular, the present invention relates to Cas effector proteins, fusion proteins comprising such proteins, and nucleic acid molecules encoding them. The invention also relates to complexes and compositions for nucleic acid editing (eg, gene or genome editing) comprising the proteins or fusion proteins of the invention, or nucleic acid molecules encoding them. The invention also relates to methods for nucleic acid editing (eg, gene or genome editing) using proteins or fusion proteins comprising the invention. Background technique [0003] CRISPR / Cas techn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/113C12N15/90C07K19/00
CPCC12N9/16C12N15/113C12N15/902C07K2319/00C12N2310/20C12N9/22C07K2319/09C07K2319/20C07K2319/40C07K2319/71C07K2319/70C12N15/90C12N15/62A61K48/00A61K47/62A61K31/7088A61K38/465C12N15/11C12N15/907C12N2800/80
Inventor 赖锦盛周英思李英男张继红王莹莹吕梦璐张湘博赵海铭宋伟彬
Owner CHINA AGRI UNIV
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