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Efficient sgRNA screening system and efficient sgRNA screening method

A technology of nucleic acid constructs and reporter genes, applied in botany equipment and methods, biochemical equipment and methods, viruses/bacteriophages, etc., can solve the problems of time-consuming and reagent consumption, inability to accurately quantify, expensive deep sequencing, etc., to achieve The effect of quick construction and easy evaluation

Active Publication Date: 2017-05-10
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The aforementioned methods all require long-term cell culture and screening operations, consuming a lot of time and reagents
Mismatch enzyme assays often have false positive cleavage and cannot be accurately quantified
Deep sequencing is expensive and requires analysis of large amounts of sequencing data

Method used

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  • Efficient sgRNA screening system and efficient sgRNA screening method
  • Efficient sgRNA screening system and efficient sgRNA screening method
  • Efficient sgRNA screening system and efficient sgRNA screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Embodiment 1, the construction of plasmid pCAG-EGFP-Luc plasmid

[0126] The pEGFP-N1 (Clontech) plasmid was linearized by Not I digestion and dephosphorylated by CIAP alkaline phosphatase; the full-length CDS of luciferase was amplified by PCR using the pGL3-Basic (Promega) plasmid as a template, and the PCR product was purified and linearized An assembly reaction (Gibson Assembly, NEB) was performed on the transformed pEGFP-N1. After heat-shock transformation of Escherichia coli, plates were spread, single clones were picked, sequenced to confirm that the sequence was completely correct, and the pCAG-EGFP-Luc plasmid was obtained, the sequence of which is shown in SEQ ID NO:1.

Embodiment 2

[0127] Embodiment 2, construction of pCAG-target-EGFP-Luc plasmid

[0128] 2.1. The artificially synthesized forward monomer DNA sequence is (5'-TCGAGATGN 1 N 2 N 3 ---N 60 -3'), and the reverse monomeric DNA sequence is (5'-TTCGA N' 60 ---N' 3 N' 2 N' 1 CAT-3'). Wherein, N'x is the reverse complementary sequence of Nx, N and N' represent base A, T, G or C, N 1 -N 60 Contains the sequences recognized by all sgRNAs.

[0129] 2.2. Anneal the above-synthesized two monomeric DNAs targeting the target gene to form a double-stranded DNA with cohesive ends.

[0130] 2.3. The pCAG-EGFP-Luc plasmid constructed in Example 1 was digested with Xho I and Hind III, linearized, mixed with double-stranded DNA, ligated with T4 DNA ligase, and then transformed into Escherichia coli competent DH5α. Extract the plasmid, that is, construct the pCAG-target-EGFP-Luc plasmid.

Embodiment 3

[0131] Example 3. Validation of the effectiveness of the sgRNA screening system by screening the high-efficiency sgRNA of the mouse RIP1 gene

[0132] 3.1. Construction of a fluorescent-luciferase dual reporter plasmid that mimics the mouse RIP1 genome—pCAG-mRIP1-EGFP-Luc plasmid

[0133] 3.1.1. Artificially synthesized forward monomer DNA sequence

[0134] (5'-TCGAGATGATGGCATCCAGTGACCTGCTGGAGAAGACAGACCTAGACAGCGGAGGCTTCGGGA-3', SEQ ID NO:2); and the reverse monomeric DNA sequence (5'-TTCGATCCCGAAGCCTCCGCTGTCTAGGTCTGTCTTCCAGCAGGTCACTGGATGCCATCAT-3', SEQ ID NO:3).

[0135] 3.1.2. Annealing the two monomer DNAs synthesized above for the target gene to form double-stranded DNA with cohesive ends;

[0136] 3.1.3. The pCAG-EGFP-Luc plasmid obtained in Example 1 was digested with Xho I and Hind III, linearized, mixed with double-stranded DNA, ligated with T4 DNA ligase, and transformed into Escherichia coli competent DH5α. The plasmid was extracted, that is, the pCAG-mRIP1-EGFP-Luc...

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Abstract

The present invention relates to an efficient sgRNA screening system and an efficient sgRNA screening method, and particularly provides a nucleic acid construct, which comprises: a first reporter gene expressing a first reporter protein; a second reporter gene expressing a second reporter protein and located at the 3' terminal of the first reporter gene; and a multiple cloning site located before the initiation codon of the first reporter gene. The present invention further provides a product, which comprises the nucleic acid construct and a nucleic acid construct expressing CRISPR-Cas9. The present invention further provides uses of the nucleic acid construct or the product in evaluation of sgRNA-mediated genome modification activity. Compared to the traditional sgRNA activity detection method, the efficient sgRNA screening method of the present invention has the following characteristics that the sgRNA activity can be evaluated quickly and easily, the sgRNA activity can be accurately quantified, the waste of labor, time and reagents is avoided, and the foundation is established for the efficient application of the CRISPR-Cas9 technology in the genome editing.

Description

technical field [0001] The invention relates to a high-efficiency sgRNA screening system and method. Background technique [0002] The existing sgRNA activity detection is to design multiple sgRNAs for the target genome sequence and clone them into the expression vector (transient expression system / lentivirus system), and then introduce them into the target cells by transfection / lentivirus infection, fluorescent protein ( GFP) / puromycin (Puro) screened positive cells to extract the genome, then PCR amplify the target genome sequence, and then use the mismatch enzyme (CEL1 or T7E1 enzyme) to cut the heterozygous double-strand that recognizes the mismatch, and the product DNA is subjected to electrophoresis. Grayscale analysis was used to evaluate sgRNA activity, and electrophoresis showed that the higher the grayscale of the cut band, the higher the activity. Alternatively, directly perform deep sequencing on the PCR-amplified target genome sequence to analyze the frequency ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N7/01C12Q1/66
Inventor 章海兵张海威
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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