A method and application of efficient deletion of large genome fragments based on CRISPR-NCAS3 system

A gene editing and amino acid technology, applied in the field of efficient deletion of large fragments of genomes, can solve the problems of lack of genome editing tools, hindering the research on the cytological properties of Zymomonas mobilis, etc.
CN113528408BActive Publication Date: 2022-03-01武汉睿嘉康生物科技有限公司
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
武汉睿嘉康生物科技有限公司
Publication Date
2022-03-01

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a method and application of a high-efficiency genome large fragment deletion method based on a CRISPR-nCas3 system. In the present invention, bioinformatics methods are firstly used to determine the essential genes to be retained and the non-essential genes that can be deleted, and select a non-essential gene with a length of 10Kb as the target knockout large fragment. Then, a spacer sequence is designed on the coding strand and the template strand of the target gene sequence, and two crRNAs transcribed and processed from the spacer sequence work together with the modified nCas3 single-stranded endonuclease to complete the double-sequence analysis of the target DNA sequence. chain breakage. Finally, the artificial CRISPR cluster and the donor DNA sequence were assembled on the carrier, and transformed into Zymomonas mobilis cells by electroporation to complete the editing.
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Description

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method and application of a high-efficiency genome large fragment deletion method based on a CRISPR-nCas3 system. Background technique

[0002] In recent years, the use of microorganisms for metabolic engineering, systems biology, and synthetic biology has made good progress. For the rational design and construction of microbial cell factories, living cells or enzymes are used to treat renewable biomass, such as It provides an important theoretical basis for the transformation of cellulose and other substances, the production of bioenergy, and the industrialization of biosmelting. Bioenergy regeneration is one of the effective means to solve the problems of resource and energy shortage and serious environmental pollution that human beings are currently facing.

[0003] CRISPR-Cas is a prokaryotic adaptive immune system widely present in most bacteria and archaea, which...

Claims

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