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Dna-cutting agent

A technology of DNA molecules and DNA sequences, which is applied in the fields of molecular biology and microbiology, can solve problems such as inability to develop and reduce acetic acid production, and achieve the effect of increasing versatility

Pending Publication Date: 2021-12-10
JOINT CO BIOCAD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To date, it has not been possible to develop efficient procedures for the production of C. cellulolyticum strains with mutations in the phosphotransacetylase and acetate kinase genes that reduce acetate production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1. Testing the activity of СсCas9 protein in cleaving various DNA targets

[0074] To examine the ability of CcCas9 to recognize various DNA sequences flanked by NNNNGNA 3' sequences, experiments were performed on in vitro cleavage of DNA targets from the human grin2b gene sequence (see Table 1 below).

[0075] Table 1. DNA targets isolated from the human grin2b gene.

[0076] DNA target PAM ctacatcacgtaacctgtct tagaAgA gaacgagctctgctgcctga cacgGcc agaacgagctctgctgcctg acacGgc acggccaaccaccaaccagaa cttgGgA tccgctctgggcttcatctt caactcg cgactccctgcaaacacaaa gaaagag atctacatcacgtaacctgt cttaGaA tatctcctttcattgagcac caaaccc

[0077] Perform in vitro DNA cleavage reactions under similar conditions to the experiments described above. As a DNA target, a fragment of the human grin2b gene with a size of about 500bp was used:

[0078]

[0079] Experimental results show that CcCas9 in complex with...

Embodiment 2

[0080] Example 2. Temperature range of CcCas9 activity

[0081] In order to determine the temperature range of the CcCas9 protein, experiments on in vitro cleavage of DNA targets were performed under different temperature conditions.

[0082] To this end, target DNA flanked by the PAM sequence GAGAGTA was subjected to cleavage at different temperatures by the CcCas9 effector complex with the corresponding guide RNA ( Figure 6 ).

[0083] The CcCas9 protein was found to have activity over a wide temperature range. Maximum nuclease activity is achieved at a temperature of 45°C, while the protein is sufficiently active in the range of 37°C to 55°C. Thus, CcCas9 in complex with guide RNA is a new tool for cleavage (double-strand break formation) in DNA molecules restricted to the sequence 5'-NNNNNGA-3', with a temperature range of 37°C to 55°C. A schematic diagram of the complex of target DNA with crRNA and tracer RNA (tracrRNA) that together form a guide RNA is shown in Fig...

Embodiment 3

[0085] from the genus Clostridium ( Clostridium ) of the closely related organism Cas9 protein. So far, only one type II CRISPR Cas system has been found in Clostridium, which is derived from Clostridium perfringens ( Clostridium perfringens) Cas9 CRISPR Cas system (Maikova A et al., New Insights Into Functions and Possible Applications of Clostridium difficile CRISPR-Cas System .Front Microbiol. 2018 Jul 31;9:1740).

[0086] The Cas9 protein from Clostridium perfringens bacteria has 36% identity with the CcCas9 protein (the degree of identity is calculated using BLASTp software, default parameters). A comparable Cas9 protein from S. aureus in size has 28% identity to CcCas9 (BLASTp, default parameters).

[0087] Thus, the CcCas9 protein differs significantly in amino acid sequence from other Cas9 proteins studied to date, including Cas9 proteins found in related organisms.

[0088] Those skilled in the field of genetic engineering will understand that the CcCas9 protei...

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Abstract

The present invention describes a novel bacterial nuclease of a CRISPR-Cas9 system from the bacterium Clostridium celluloliticum, as well as the use of said nuclease for creating strictly specific two-strand cuts in a DNA molecule. The present nuclease exhibits unusual qualities and can be used as an instrument for introducing changes into 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 in order to cut genomic and plasmid DNA in a large number of specific places and at wide ranges of temperatures. The invention also simplifies editing the genome of the biotechnologically relevant bacterium Clostridium cellulolyticum.

Description

technical field [0001] The present invention relates to the fields of molecular biology and microbiology, in particular it discloses new bacterial nucleases of the CRISPR-Cas system. The present invention can be used as a tool for strictly specific modification of DNA in various 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 into the DNA sequence. 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 comes to the CRISPR-Cas system, the structure th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/55C12N15/113C12N15/90
CPCC12N9/22C12N2310/20C12N15/102C12Q1/68C12N15/111C12N15/907C12N2800/80
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 JOINT CO BIOCAD
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