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Nucleic acid molecule targeting CYP4V2 gene mutation site and application thereof

A nucleic acid molecule and gene technology, applied in the field of gRNA and donor nucleic acid molecules, can solve problems such as limited curative effect

Active Publication Date: 2021-07-23
CHIGENOVO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This approach can only partially restore the function of mutant cells and has limited efficacy

Method used

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  • Nucleic acid molecule targeting CYP4V2 gene mutation site and application thereof
  • Nucleic acid molecule targeting CYP4V2 gene mutation site and application thereof
  • Nucleic acid molecule targeting CYP4V2 gene mutation site and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Example 1 Design sgRNA

[0110] 1. Screening of sgRNA

[0111] According to the DNA sequence of the CYP4V2 gene, the PAM sequence in the intron region between exon 6 and exon 7 of CYP4V2 is designed as NNGRRT and NNGRR (Staphylococcus aureus, Staphylococcus aureus, SA; SaCas9), with a length of 21bp sgRNA.

[0112] According to the scores, a total of 7 sgRNAs (5 for NNGRRT and 2 for NNGRR) were designed, and their sequences are shown in Table 1.

[0113] Table 1. CYP4V2-HITI method benchling online design of sgRNA

[0114]

[0115]

[0116] 2. sgRNA synthesis

[0117] According to the restriction site Bbs1 sequence, add the Bbs1 restriction site in the upstream and downstream of the designed sgRNA; and design corresponding primers within 400 bp upstream and downstream of each sgRNA. The corresponding oligonucleotide sequences and primers were designed in Table 2.

[0118] Table 2 Design of sgRNA

[0119]

[0120]

[0121] 3. The specific steps of sgRNA...

Embodiment 2

[0232] Example 2 Verifying the impact of sgRNA targets on cleavage

[0233] 1. The four sgRNAs correspond to the pMD19-T minigene plasmid, and its control negative plasmid and positive plasmid design.

[0234] According to the donor HITI method, the characteristics of PAM+3bpsgRNA / 18bp sgRNA fragments will remain after insertion, and minigene1.2.3.4 is designed to correspond to sgRNA1.2.3.4. The minigene fragments are as follows Figure 4 shown.

[0235] Second, the minigene plasmid construction steps are as follows

[0236] 1. Synthesize minigene DNA fragments and positive control (normal wild-type intron, which does not affect cleavage) and negative control (patient mutated intron, which affects cleavage) DNA fragments.

[0237] 2. Restriction plasmid vector

[0238] The plasmid vector is PMD-19T-MCS, see the plasmid map Figure 5 .

[0239] (1) Digest the plasmid with KpnI enzyme and MluI

[0240] In CutSmart buffer, the KpnI enzyme and MluI enzyme were incubated with...

Embodiment 3

[0317] Embodiment 3 Design and screening of Donor

[0318] 1. Carrier construction:

[0319] The sequence between intron 6 and exon 7-11 of the CYP4V2 wild-type gene was used as the Donor sequence; the Donor sequence and the EGFP reporter gene were constructed together into the pX601 vector (pX601-sgRNA1 to pX601-sgRNA4 of sgRNA1-4 carrier) to obtain the pX601-donor(1-4)-EGFP carrier; the sgRNA carrier uses the pX601-sgRNA(1-4) carrier described in Example 1. PX601 plasmid map as figure 1 shown.

[0320] Wherein, the length of intron 6 is adjusted according to the sgRNA cutting site, the Donor sequence of exons 7-11 is shown in SEQ ID NO:38; the EGFP sequence is shown in SEQ ID NO:39.

[0321] 2. Transfection of iPSCs by PEI method

[0322] 1. The specific steps are as follows:

[0323] Take the sgRNA1 group as an example:

[0324] (1) Take four 6-well plates, inoculate iPSC cells, and perform transfection when the cell density grows to 80%;

[0325] (2) The first group...

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Abstract

The invention relates to a gRNA (guide Ribonucleic Acid) targeting a CYP4V2 gene and a donor nucleic acid molecule containing a CYP4V2 gene segment, and further relates to application of the gRNA and the donor nucleic acid molecule in diseases.

Description

technical field [0001] This application relates to the field of biomedicine, in particular to a gRNA and a donor nucleic acid molecule for treating CYP4V2 gene mutation diseases. Background technique [0002] (Bietti crystalline dystrophy, BCD), also known as crystalline retinitis pigmentosa, crystalline corneal retinal degeneration (Bietti Crystalline Corneoretinal Dystrophy), crystalline retinopathy (Bietti Crystalline Retinopathy), Bietti's retinal degeneration sRetinal Dystrophy) (OMIM 210370)) is a blinding autosomal recessive retinal degenerative disease. The CYP4V2 gene is one of the BCD pathogenic genes discovered so far (Li et al., Am J Hum Genet. 74:817-826, 200). CYP4V2 (cytochrome P450, family 4, subfamily V, polypeptide 2, (0MIM 608614), synonym: CYP4AH1) belongs to the cytochrome P450 superfamily and is a heme-thiolate protein cytochrome P450 subfamily 4 (CYP4 )a member of. [0003] At present, there are many methods for the treatment of this disease, such a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/864C12N5/10A61K48/00A61K31/7088A61P27/02
CPCC12N15/1137C12N15/86C12N9/0079A61K48/005A61K48/0008A61K48/0075A61K31/7088A61P27/02C12Y114/15004C12N2310/20C12N2750/14143C12N9/0071C12N15/52A01K2207/15A01K2227/105A01K2217/072C12Y114/13
Inventor 杨丽萍孟祥陈邵宏和赛超张凡史天永曾露颖卢化
Owner CHIGENOVO CO LTD
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