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

Optimum design method of structure-based CRISPR protein

An optimized design and protein technology, applied in the field of protein engineering, can solve problems such as time-consuming, labor-intensive, blindness, etc.

Active Publication Date: 2020-11-06
FUDAN UNIV
View PDF3 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods can help people obtain new mutants of Cas9, these methods are relatively blind, time-consuming and labor-intensive

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
  • Optimum design method of structure-based CRISPR protein
  • Optimum design method of structure-based CRISPR protein
  • Optimum design method of structure-based CRISPR protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0048] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0049] The materials and reagents used in the following examples are obtained from commercial sources unless otherwise specified.

[0050] 1. SpCas9 combinatorial mutation design

[0051] Step 1: Identify the amino acid positions that have an important impact on the properties of Cas9.

[0052] The seven directed evolution sites (A262T, R324L, S409I, E480K, E543D, M694I, and E1219V) of xCas9 mutations are distributed on both sides of the sgRNA-DNA heteroduplex (attached figure 2 ). These seven amino acids work together to expand the PAM recognition range of xCas9 and reduce its off-target effects. Therefore, these 7 sites were selected as the amino acid sites for Rosetta optimization design.

[0053] Step 2: Minimize Relax energy.

[0054] The SpCas9 structure (PDB ID: 4UN3) was iterated for 25 rounds with the Relax module, and the structure with the lowe...

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 technical field of protein engineering, and specifically relates to an optimum design method of a structure-based CRISPR protein. The optimum design method comprises the following steps: carrying out analysis and comparison based on an analyzed CRISPR protein structure, so as to find out an amino acid site greatly influencing a certain function of Cas9; and optimizinga Protein-DNA interaction interface of the Cas9 through combination with Rosetta, finally carrying out designing to successfully obtain a novel mutant yCas9. The mutant has shearing activities equal to xCas9: the mutant has a wide PAM recognition range, is capable of recognizing NGG, NGA, NGT, NGC, GAA and GAT and is low in missing rate during gene editing. Therefore, the yCas9 functionally keepspace with the xCas9 and is a gene editing tool with a potential application value, thereby proving the validity and practicability of the design method.

Description

technical field [0001] The invention belongs to the technical field of protein engineering, and specifically relates to a structure-based optimal design method of CRISPR protein, and also relates to a novel mutant of SpCas9 obtained by the optimal design method, and its application as a gene editing tool. Background technique [0002] CRISPR is the immune system against foreign nucleic acid invasion in bacteria and archaea. In recent years, it has been developed as a gene editing tool that specifically cuts DNA fragments [1] . The most commonly used type II CRISPR system is Streptococcus pyogenes Cas9 (SpCas9) [2] . However, SpCas9 has the problems of limited recognition range of PAM (protospacer adjacent motifs) and easy off-target during gene editing, which limits its wider application [3-5] . Therefore, only by expanding the recognition range of PAM and reducing off-target effects can SpCas9 technology play a greater role in the field of gene editing. [0003] In or...

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
IPC IPC(8): C12N9/22C12N15/55C12N15/70C12N1/21C12N15/113C12R1/19
CPCC12N9/22C12N15/113C12N15/70C12N2310/20
Inventor 黄强薛冬梅朱海霞杜文豪
Owner FUDAN 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