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
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Although these methods can help people obtain new mutants of Cas9...

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  • 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

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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...

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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

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Application Information

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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
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