Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Novel CRISPR/Cas12f enzymes and systems

A C-terminal and protein technology, applied in the field of nucleic acid molecules, Cas effector proteins, nucleic acid editing complexes and compositions, can solve the problem of reducing off-target effects and achieve the effect of reducing off-target effects

Active Publication Date: 2021-05-25
CHINA AGRI UNIV
View PDF5 Cites 16 Cited by
  • 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 a strict 5'-TTN, so its target site is easier to predict than other systems, thereby reducing potential off-target effects

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Novel CRISPR/Cas12f enzymes and systems
  • Novel CRISPR/Cas12f enzymes and systems
  • Novel CRISPR/Cas12f enzymes and systems

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0216]The acquisition of embodiment 1.Cas12f gene and Cas12f guide RNA

[0217] 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.

[0218] 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 when mining CRISPR effector proteins.

[0219] 3. Acquisition of CRISPR-related macromolecular proteins: Extend each CRISPR locus upstream and downstream by 10Kb, and identify non-redundant macromolecular proteins in the adjacent C...

Embodiment 2

[0224] Example 2.Cas12f gene processing of mature crRNA

[0225] 1. Artificially synthesize the double-stranded DNA molecule shown in SEQ ID NO:4, and simultaneously artificially synthesize the double-stranded DNA molecule shown in SEQ ID NO:10.

[0226] 2. Ligate the double-stranded DNA molecule synthesized in step 1 with the prokaryotic expression vector pACYC-Duet-1 to obtain the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f.

[0227] The recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f was sequenced. Sequencing results show that the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f contains the sequences shown in SEQ ID NO:4 and SEQ ID NO:10, and expresses the Cas12f.4 protein and SEQ ID NO:1 shown in SEQ ID NO:1 Cas12f.4 prototype direct repeat sequence shown in NO:7. The recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f was introduced into Escherichia coli EC100 to obtain a recombinant bacterium, which was named EC100-CRISPR / Cas12f.

[0228] 3. Take a single clone of EC100-C...

Embodiment 3

[0239] PAM domain identification of embodiment 3.Cas12f gene

[0240] 1. Construct the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f and sequence it. According to the sequencing results, the structure of the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f is described as follows: replace the small fragment between the recognition sequences of the restriction endonuclease Pml I and Kpn I of the vector pACYC-Duet-1 with SEQ ID NO : A double-stranded DNA molecule shown at positions 1 to 3713 from the 5' end in the sequence shown in 4. The recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f expresses the Cas12f.4 protein shown in SEQ ID NO:1 and the Cas12f guide RNA shown in SEQ ID NO:25.

[0241] 2. An expression cassette is contained in the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f, and the nucleotide sequence of the expression cassette is shown in SEQ ID NO:23. In the sequence shown in SEQ ID NO:23, the 1st to 44th from the 5' end is the nucleotide sequence of the pLacZ promo...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the field of nucleic acid editing, in particular to the technical field of regularly clustered interspaced short palindromic repeats (CRISPR). Specifically, the present invention provides Cas effector proteins, fusion proteins comprising such proteins, and nucleic acid molecules encoding them, and also provides complexes for nucleic acid editing (for example, gene or genome editing) comprising the above-mentioned proteins or nucleic acid molecules. Objects and compositions, and methods for nucleic acid editing (eg, gene or genome editing) comprising the above proteins.

Description

technical field [0001] 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 [0002] CRISPR / Cas technology is a widely used gene editing technology. It uses RNA guidance to specifically bind to target sequences on the genome and cuts DNA to generate double-strand breaks. It uses biological non-homologous end joining or homologous ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C07K19/00C12N15/113C12N15/90C12N9/16
CPCC12N9/16C12N15/113C12N15/902C07K2319/00C12N2310/20C12N15/90C12N9/22C07K2319/09C07K2319/01C07K2319/40C07K2319/71C07K2319/70C12N15/111C12N15/62
Inventor 赖锦盛周英思朱金洁易飞张湘博赵海铭宋伟彬
Owner CHINA AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com