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Dna-cutting agent

A technology of DNA molecules and DNA sequences, applied in the field of new Cas nucleases, which can solve problems such as restricting the use of nucleases

Pending Publication Date: 2021-11-05
BIOCAD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Various CRISPR-Cas proteins have different PAM sequences that limit the potential to use nucleases at any DNA region

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1. Testing the activity of DfCas9 protein in cleaving various DNA targets

[0064] To test the ability of DfCas9 to recognize different DNA sequences flanked by the motif 5'-NN(G / A)NA(C / T)N-3', experiments with in vitro cleavage of DNA targets from the human grin2b gene sequence were performed ( See Table 1).

[0065] Table 1. DNA targets isolated from the human grin2b gene.

[0066] DNA target PAM attttctctcattctgcaga gcaaata tctaagacaggttacgtgat gtagatc aatgaaaggagataaggtcc ttgaatt caccttttattgccttgttc aaggatt ggcattgctgtcatccctcgt gggcact ttgcagtatctagcctcttc taagaca

[0067] Perform in vitro DNA cleavage reactions under similar conditions to previous experiments. Guide crRNAs were synthesized for each target sequence. A fragment of the human grin2b gene with a size of approximately 760 bp was used as the DNA target:

[0068] .

[0069] A sequence flanked from the 3' end by a DNA region correspo...

Embodiment 2

[0071] Example 2. Effect of Mn2+ ion concentration on nuclease functionality

[0072] To test the effect of Mn2+ ions on DfCas9 nuclease activity, experiments were performed in vitro to cleave double-stranded DNA fragments containing target DNA sequences restricted to the PAM sequence AAAAAC with the consensus sequence 3'-NN(G / A) NA(C / T)-5'. Reactions were performed using 1x CutSmart Buffer (NEB), target DNA at a concentration of 20 nM, and trRNA / crRNA at a concentration of 2 μM.

[0073] As a DNA target, we use

[0074] .

[0075] React using tracrRNA:

[0076] and

[0077] .

[0078] Figure 7 It was shown that increasing the concentration of MnCl2 from 5 mM to 10 mM resulted in more efficient cleavage of the DNA target, whereas further increases in the concentration of divalent ions did not affect the reaction efficiency. Therefore, efficient DfCas9 activity requires the presence of manganese ions at a concentration of 10 mM or higher.

Embodiment 3

[0079] Example 3. Use of hybrid guide RNAs for cleavage of DNA targets

[0080] sgRNA is a form of guide RNA that is a fusion of tracrRNA (tracer RNA) and crRNA. To select the optimal sgRNA, we constructed three variants of this sequence that differ in the length of the tracrRNA-crRNA duplex. RNA was synthesized in vitro, and experiments were performed on it to cleave DNA targets ( Figure 8 Reactions for DNA cleavage by DfCas9 using various sgRNA variants are shown).

[0081] The selected sgRNA3 was as efficient as native tracrRNA and crRNA sequences, cleaving more than 50% of the DNA target.

[0082] While modifying the sequence that pairs directly with the DNA target, this sgRNA variant can be used to cleave any other target DNA.

[0083] The following RNA sequences were used as hybrid RNAs:

[0084] 1-sgRNA1 24DR:

[0085]

[0086] 2 - sgRNA2 18DR

[0087]

[0088] 3 - sgRNA3 30DR

[0089]

[0090] 4 - Control (without RNA)

[0091] 5 - Positive control (c...

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Abstract

The present invention describes a novel bacterial nuclease of a CRISPR-Cas9 system from the bacteria Defluviimonas sp. 20V17, as well as the use of said nuclease for creating strictly specific two-strand cuts in a DNA molecule. The present nuclease possesses unusual properties and can be used as an instrument for introducing changes at strictly specified places in a genomic DNA sequence of single-celled and multi-celled organisms. The invention thus increases the universality of accessible CRISPR-Cas9 systems, which makes it possible to use Cas9 nuclease from various organisms to cut genomic or plasmid DNA in a large number of specific sites and under various conditions.

Description

technical field [0001] The present invention relates to biotechnology for cutting DNA and editing the genomes of various organisms, in particular new Cas nucleases for the CRISPR-Cas system. The technique could one day be used for gene therapy of inherited human diseases, as well as for editing the genomes of other organisms. Background technique [0002] The modification of DNA sequence is one of the hot issues in the field of biotechnology today. Editing and modifying the genomes of eukaryotes and prokaryotes, as well as manipulating DNA in vitro, requires the targeted introduction of double-strand breaks within the DNA sequence. [0003] To solve this problem, the following technologies are currently used: an artificial nuclease system containing a zinc finger domain, a TALEN system, and a bacterial CRISPR-Cas system. The first two techniques require laborious optimization of nuclease amino acid sequences for recognition of specific DNA sequences. By contrast, when it ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12N2310/20C12N15/102C12N9/22C12Q1/68C12N15/111
Inventor K·V·谢韦里诺夫S·A·什马科夫D·N·阿塔莫诺娃I·I·高亚宁O·S·穆沙罗娃I·V·皮斯库诺娃I·V·费多罗娃T·I·祖布科M·A·霍多尔科夫斯基G·E·波贝加洛夫A·N·阿尔谢尼涅夫P·A·赛尔科娃A·A·瓦西列娃T·O·阿塔莫诺娃M·V·阿布拉莫娃
Owner BIOCAD
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