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SgRNA specifically targeting human RSPO2 gene in CRISPR-Cas9 system and activation method and application thereof

A specific, genetic technology that can be used in retro-RNA viruses, DNA/RNA fragments, other methods of inserting foreign genetic material, etc., and can solve problems such as adverse biological effects

Active Publication Date: 2018-07-06
JIAXING NO 1 HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Wnt signaling pathway is involved in a variety of biological processes, including differentiation and maintenance of cell morphology and function, immunity, cell carcinogenesis and apoptosis, and directly blocking this signaling pathway may have a wide range of adverse biological effects

Method used

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  • SgRNA specifically targeting human RSPO2 gene in CRISPR-Cas9 system and activation method and application thereof
  • SgRNA specifically targeting human RSPO2 gene in CRISPR-Cas9 system and activation method and application thereof
  • SgRNA specifically targeting human RSPO2 gene in CRISPR-Cas9 system and activation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 sgRNA sequence design

[0085] There are currently no clear principles for sgRNA design to ensure efficient and specific activation of target gene (CRISPRa) expression. According to our previous work and experience, the design of the sgRNA sequence in the present invention is as follows: (1) the length of the sgRNA is 20 base sequences; (2) the target site of the sgRNA that specifically activates the human RSPO2 gene is located on the RSPO2 gene. (3) choose to design the target at -120~-75 upstream of the RSPO2 gene transcription initiation site; (4) in the RSPO promoter region, select PAM as 5'-NGG; (5) to make the vector The U6 promoter is valid, so ensure that the sgRNA target sequence starts with G; (6) The format of the sgRNA target sequence is:

[0086] 5`-G-(19N)-NGG-3`(sgRNA target sequence starts with G)

[0087] Or 5`-(20N)-NGG-3`(sgRNA target sequence does not start with G)

[0088] In the above target sequence format, 19N or 20N represents the...

Embodiment 2

[0090] Example 2 sgRNA sequence selection

[0091]Use Blast (www.ncbi.nlm.nig.gov / Blast) to perform homology analysis on the candidate sgRNA sequence and the genome database to ensure that the target sequence of the designed sgRNA is unique and will not be homologous to other gene sequences except the human RSPO2 gene . At the same time, the sgRNA was screened according to the following principles to obtain the sgRNA sequence that efficiently and specifically activates the human RSPO2 gene: (1) the sgRNA target is located at the DNase hypersensitive site; (2) the sgRNA target is located at the transcription initiation site of the RSPO2 gene Upstream -120~-75; (3) Low off-target rate

[0092] According to the above method, among the 10 sgRNAs targeting the human RSPO2 gene, only 3 sgRNA sequences (corresponding to SEQ ID NO. , 6), the target sequence of the sgRNA and the corresponding PAM sequence are shown in Table 1 (corresponding to SEQ ID NO.1, 3, 5 in the sequence list...

Embodiment 3

[0093] Example 3 sgRNA oligonucleotide synthesis

[0094] For the above sgRNA sequence targeting human RSPO2 gene, add BsmBI restriction site and PAM sequence at both ends: (1) According to the selected sgRNA, add CACC (BsmBI restriction site sticky Complementary sequence at the end) and G (to ensure that the U6 promoter is effective) to obtain a forward oligonucleotide; (2) According to the selected sgRNA, obtain the complementary strand of DNA corresponding to its target sequence, and add AAAC at its 5' end ( The complementary sequence of the cohesive end of the BsmBI restriction site), add C at the 3' end to get the reverse oligonucleotide

[0095] The above-mentioned forward oligonucleotide and reverse oligonucleotide were respectively synthesized by chemical synthesis method, and the obtained oligonucleotide sequences are shown in Table 2.

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Abstract

The invention discloses a sgRNA specifically targeting human RSPO2 gene in a CRISPR-Cas9 system and an activation method and application thereof, and belongs to the field of biotechnology. The CRISPR-Cas9 system specifically activates the expression of the human RSPO2 gene, and introduces the activity of human hepatic stellate cells to activate Wnt signaling pathway so as to significantly upregulate the expression of liver fibrosis markers alpha-SMA and Collagen I. The CRISPR-Cas9 system designed for RSPO2 gene targets can effectively activate the hepatic stellate cells, thereby providing an effective way for liver fibrosis research.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an sgRNA specifically targeting the human RSPO2 gene in a CRISPR-Cas9 system and an activation method and application thereof. Background technique [0002] Clustered regularly interspaced short palindromic repeat system (CRISPR-Cas9) widely exists in bacteria and archaea, and is an RNA-mediated, heritable acquired immune system. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) consists of highly conserved repeats (repeats) and multiple different spacers (spacers) arranged in sequence. The length of the repeat sequence is usually 21-48bp, and the repeat sequence is separated by a 26-72bp spacer sequence separated. Cas9 (CRISPR associated) is a double-stranded DNA nuclease with two domains: ①HNH-like domain cuts the DNA strand complementary to crRNA (CRISPR RNA), ②RuvC-like domain cuts the non-complementary strand. The basic mechanism of CRISPR-Cas9 is:...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C12N15/90
CPCC07K14/4702C12N9/22C12N15/113C12N15/86C12N15/907C12N2310/10C12N2740/15043C12N2800/107C12N2810/10C12N15/1138C12N2330/51C12N2310/20C12N15/11
Inventor 虞玲华姚明
Owner JIAXING NO 1 HOSPITAL
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