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Nicking enzyme and application thereof to genome base substitution

A technology of cutting enzymes and plant genomes, applied in genetic engineering, enzymes, hydrolytic enzymes, etc., can solve problems such as difficult removal

Active Publication Date: 2019-01-25
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although plants are different from animals, off-target sites can be removed by genetic segregation in offspring, but because some potential off-target sites may be unknown, it is difficult to remove them purposefully in offspring

Method used

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  • Nicking enzyme and application thereof to genome base substitution
  • Nicking enzyme and application thereof to genome base substitution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1, HypaCas9n&PmCDA1&UGI system C T base substitution efficiency

[0036] 1. Construction of genome editing vector

[0037] 1. SpCas9n&PmCDA1&UGI vector: artificially synthesized circular plasmid shown in sequence 1 of the sequence listing.

[0038] Sequence 1 of the sequence listing includes the following three expression cassettes:

[0039] Sequence 1 from the 131st-1283rd position of the 5' end is the expression cassette I, wherein the 131-467th position is the nucleotide sequence of the OsU3 promoter, the 474-550th position is the nucleotide sequence of the pre-tRNA, and the 1st position is the nucleotide sequence of the pre-tRNA. 551-570 is the nucleotide sequence of CS650 target, 571-646 is the nucleotide sequence of sgRNA, 647-723 is the nucleotide sequence of pre-tRNA, 724-743 is the CS651 target The nucleotide sequence of the point, the 744-819th is the nucleotide sequence of the sgRNA, the 820-896th is the nucleotide sequence of the pre-tRNA, and the 89...

Embodiment 2

[0062] Example 2, HypaCas9n&PmCDA1&UGI system off-target effect

[0063] 1. Construction of genome editing vector

[0064] SpCas9n&PmCDA1&UGI-T1: A circular plasmid obtained by replacing sequence 1 of the sequence listing from position 551-916 of the 5' end with sequence 3 of the sequence listing.

[0065] SpCas9n&PmCDA1&UGI-T2: A circular plasmid obtained by replacing sequence 1 of the sequence listing with sequence 4 of the sequence listing from 5' end 551-916.

[0066] SpCas9n&PmCDA1&UGI-T3: A circular plasmid obtained by replacing the sequence 1 of the sequence listing from the 551-916 position of the 5' end with the sequence 5 of the sequence listing.

[0067] SpCas9n&PmCDA1&UGI-T4: A circular plasmid obtained by replacing sequence 1 of the sequence listing with sequence 6 of the sequence listing from positions 551-916 at the 5' end.

[0068] SpCas9n&PmCDA1&UGI-T5: A circular plasmid obtained by replacing the sequence 1 of the sequence listing from the 551-916 position ...

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Abstract

The invention discloses a nicking enzyme and application thereof to genome base substitution. A base editing system is constructed by fusing a nicking enzyme HypaCas9n and PmCDA1 and UGI for the firsttime, and the invention finds out that when HypaCas9n&PmCDA1&UGI is compared with SpCas9n&PmCDA1&UGI, the off-target efficiency can be reduced under the condition that C.T base substitution efficiency is basically not influenced.

Description

technical field [0001] The invention relates to a nickase and its application in genome base replacement. Background technique [0002] The emergence and development of CRISPR-Cas9 (the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) technology has become a powerful means of genome editing and has been widely used in many tissues and cells. The CRISPR / Cas9protein–RNA complex locates on the target through the guide RNA (guide RNA), cuts and generates a DNA double-strand break (DSB, dsDNAbreak), and then, the organism will instinctively initiate a DNA repair mechanism to repair the DSB. Generally, there are two repair mechanisms, one is non-homologous end joining (NHEJ, non-homologous endjoining), and the other is homologous recombination (HDR, homology-directed repair). Usually, NHEJ repair accounts for the majority, therefore, The random indels (insertions or deletions) generated by the repair are much higher than the exact repair. Fo...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/82C12N9/22
CPCC07K2319/00C12N9/22C12N9/78C12N15/8213C12Y305/04001
Inventor 杨进孝杨永星吕欣欣赵思冯峰
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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