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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.

Active Publication Date: 2022-03-01
武汉睿嘉康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The lack of efficient and easy-to-operate genome editing tools has seriously hindered the study of the cytological properties of Zymomonas mobilis, which has good microbial cell factory characteristics, and the effective use of its advantages in bioenergy production

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Preparation and function of protein and its coding gene.

[0070] nCas3 single-stranded endonuclease refers to the introduction of mutations in the helicase functional domain of the wild-type Cas3 protein, making it lose the helicase activity, thereby transforming into nCas3 protein with only single-stranded nuclease activity. The endonuclease activity can act on a single strand of the target DNA duplex by endonucleating 1,4-phosphodiester bonds.

[0071] The amino acid sequence of the wild type (wide type) Cas3 protein involved in this embodiment of the present invention is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. Three kinds of nCas3 single-stranded endonucleases with different sequences are specifically provided in this embodiment, named K458A respectively (the lysine at position 458 of the wild-type Cas3 protein is replaced with alanine, the amino acid sequence is the same as that of SEQ ID NO .1, the difference is only t...

Embodiment 2

[0094] Example 2 Using circular DNA as a substrate to verify protein function

[0095] The pL2R plasmid (Zheng et al, 2019) was selected as the reaction substrate, and the total length of the plasmid was 3283bp. The reaction system contains 150ng pL2R circular plasmid; 2mM MgCl2; 0.5mM ATP; 250nM Cascade protein; 250nM one of the above purified Cas3 or nCas3 protein variants; 32nt crRNA. The above crRNA was synthesized by GenScript Biotechnology Co., Ltd., and its sequence is shown in SEQ ID NO.5. After reacting at 30°C for 15, 30, and 60 minutes respectively, the reaction products were run on agarose gel electrophoresis.

[0096] The result is as figure 2 as shown, figure 2 The leftmost lane in the center represents nucleic acid molecular weight standards (5.0, 3.0, 2.0kb); the rightmost Reaction time axis represents the reaction duration (15, 30, 60min); the right OC, L, SC represent circular DNA molecules after the reaction state, [OC (open circle); L (linear); SC (n...

Embodiment 3

[0097] Embodiment 3 comprises the preparation of the engineering bacterium of nCas3 single-stranded endonuclease

[0098] In this example, D608A was taken as an example to prepare engineering bacteria containing nCas3 single-stranded endonuclease.

[0099] 1. Design and construct single base editing plasmid

[0100] (1) According to the experimental requirements, it was decided to use the endogenous I-F type CRISPR-Cas editing system in the ZM4 strain to edit the target site on the genome.

[0101] Such as image 3As shown, through careful analysis of the Cas3 protein coding gene on the genome of the ZM4 strain, it was found that there was a 5'-TCC-3' PAM sequence in the cas3 gene sequence, so the 32-nt sequence downstream of it was designed as a protospacer sequence . In order to prevent the CRISPR-Cas system from destroying the transferred donor plasmid, and to complete the in situ replacement of 676,661 adenine nucleotides to cytosine nucleotides in the ZMO0681 gene, a t...

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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.

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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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/55C12R1/01
CPCC12N9/22C12N15/74
Inventor 彭文舫郝怡乐
Owner 武汉睿嘉康生物科技有限公司
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