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CRISPR-Cas9 system used for assembling DNA and DNA assembly method

A component, reporter gene technology, applied in the field of synthetic biology, can solve problems such as enhancing the rate of homologous recombination and improving the efficiency of synthetic genome assembly

Inactive Publication Date: 2016-08-03
SHENZHEN HUADA GENE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, domestic CRISPR-related patent technologies focus on gene knockout and knockout library construction, such as "Method for constructing eukaryotic gene knockout library using CRISPR / Cas9 system" (CN103668472A, 2014.03.26), and have not yet used CRISPR technology Enhanced homologous recombination rate to improve the efficiency of synthetic genome assembly related patent technology reports

Method used

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  • CRISPR-Cas9 system used for assembling DNA and DNA assembly method

Examples

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Effect test

Embodiment 1

[0099] 1. Design CRISPR sites for the reporter genes LEU2 and URA3, and the fragment replacement test gene YBR157C (see the above "CRISPR site design" section for specific methods) (Table 3).

[0100] table 3

[0101]

[0102]

[0103] 2. Acquisition and transformation of Cas9 gene expression plasmid

[0104] a. The original Cas9 gene expression plasmid pYP003 was donated by Mr. Dai Junbiao of Tsinghua University, and can also be obtained through commercial purchase or synthesized by a gene synthesis company. The sequence of Cas9 is shown as SEQ ID NO: 12 in the sequence listing.

[0105] b. Since the defective marker of the original Cas9 gene expression plasmid is LEU2, which conflicts with the reporter gene of the replacement fragment, the pYP003 plasmid was digested with restriction enzymes NheI and SpeI (NEB) to obtain the Cas9 gene fragment. Prepare the following enzyme digestion system (Table 4) in a 200 μL PCR tube, and react for 3 hours at the optimum temperatu...

Embodiment 2

[0219] The artificially synthesized chromosome or the artificially synthesized genome is extracted from the Saccharomyces cerevisiae containing the artificially synthesized chromosome or the artificially synthesized genome obtained in Example 1 by using the chromosome or genome extraction technique known in the art.

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Abstract

The invention discloses a CRISPR-Cas9 system used for assembling DNA and a DNA assembly method. The CRISPR-Cas9 system includes the following parts: a plasmid used for expressing Cas9 gene, a first guide RNA, and / or a plasmid used for expressing the first guide RNA, wherein the first guide RNA has a CRISPR site. The CRISPR site is combined with a first reporter gene carried by a semisynthetic chromosome used for assembling according to base complementation pairing rule. The CRISPR-Cas9 system has advantages of high replacement success rate of homologous recombination, less species of guide RNA which needs to design and use, less harmful effect subjected by the genomic sequence, and low off-target rate.

Description

technical field [0001] The invention relates to synthetic genome technology in the field of synthetic biology, in particular to a CRISPR-Cas9 system for DNA assembly and a DNA assembly method. Background technique [0002] CRISPR-Cas9 is a self-defense system of bacteria and archaea, which can recognize foreign double-stranded DNA through single-stranded RNA, and generate double bond breaks on it with the help of Cas9 nuclease to eliminate foreign DNA infection. The CRISPR-Cas9 system is portable and can perform DNA recognition and excision functions in cells including eukaryotes such as Saccharomyces cerevisiae after modification, and has been transformed into an excellent genome editing tool. Compared with Zinc-Finger and TALE technologies, CRISPR-Cas9 has become one of the most popular research fields because of its simple DNA recognition principle (using RNA binding recognition, unlike the former two using proteins that can bind DNA). One research direction of CRISPR-Ca...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/10C12N15/11C12N1/19C12R1/865
CPCC12N15/102C12N15/11C12N15/81C12N2310/10C12N2800/102C12N2800/80C12N2810/10C12N1/185C12R2001/865
Inventor 龚剑辉范楚瑶陈泰邓洋王云沈玥
Owner SHENZHEN HUADA GENE INST
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