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CRISPR/Cas9 system causing congenital deafness and application of CRISPR/Cas9 system in preparation of model pig nuclear donor cells

A hereditary deafness, cell technology, applied to genetically modified cells, bone/connective tissue cells, cells modified by introducing foreign genetic material, etc., can solve the problems of unclear physiological effects and deafness-causing mechanism, and reduce work costs Quantity, good applicability

Pending Publication Date: 2021-03-19
NANJING KGENE GENETIC ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

TMC1 is an essential component of hair cells in the inner ear to detect acoustic stimulation, but its physiological role and mechanism of deafness are still unclear. Pathogenesis and treatment options

Method used

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  • CRISPR/Cas9 system causing congenital deafness and application of CRISPR/Cas9 system in preparation of model pig nuclear donor cells
  • CRISPR/Cas9 system causing congenital deafness and application of CRISPR/Cas9 system in preparation of model pig nuclear donor cells
  • CRISPR/Cas9 system causing congenital deafness and application of CRISPR/Cas9 system in preparation of model pig nuclear donor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1, the preparation of plasmid

[0078] 1.1 Preparation of plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO (plasmid pKG-GE3 for short)

[0079] The original plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (referred to as plasmid pX330, the sequence is shown in SEQ ID NO: 1. The structure diagram of plasmid pX330 is shown in figure 1 . In SEQ ID NO: 1, the 440-725 nucleotides form the CMV enhancer, the 727-1208 nucleotides form the chickenβ-actin promoter, and the 1304-1324 nucleotides encode the SV40 nuclear localization signal (NLS ), the 1325-5449th nucleotide encodes the Cas9 protein, and the 5450-5497th nucleotide encodes the nucleoplasmin nuclear localization signal (NLS).

[0080] Plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, referred to as plasmid pKG-GE3, the nucleotide is shown in SEQ ID NO:2. The build method is as follows:

[0081] (1) Remove redundant and invalid sequences in the gRNA backbone

[0082] Plasmid pX330 was digested with BbsI and XbaI, and ...

Embodiment 2

[0093] Embodiment 2, the effect comparison of plasmid pX330 and plasmid pKG-GE3

[0094] Select high-efficiency gRNA targets located at the RAG1 gene:

[0095] Target of RAG1-gRNA4: 5'-AGTTATGGCAGAACTCAGTG-3' (SEQ ID NO: 9).

[0096] Primers used to amplify and detect target-containing fragments are as follows:

[0097] RAG1-nF126: 5'-CCCCATCCAAAGTTTTTAAAGGA-3' (SEQ ID NO: 10);

[0098] RAG1-nR525: 5'-TGTGGCAGATGTCACAGTTTAGG-3' (SEQ ID NO: 11)

[0099] Primary pig fibroblasts were prepared from newborn Congjiangxiang pigs (female, blood type AO).

[0100] 1. Preparation of recombinant plasmids

[0101] The plasmid pKG-U6gRNA was taken, digested with restriction endonuclease BbsI, and the vector backbone (a large linear fragment of about 3 kb) was recovered. RAG1-4S and RAG1-4A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with cohesive ends. The double-stranded DNA molecule with cohesive ends and the vector backbone were lig...

Embodiment 3

[0123] Example 3, Target Screening for TMC1 Gene Knockout

[0124] Pig TMC1 gene information: encodes ransmembrane channel like 1 protein; located on pig chromosome 1;

[0125] GeneID is 100516949, Sus scrofa. The protein encoded by the porcine TMC1 gene is shown in GENBANK ACCESSION NO.XP_020920511.1 (linear MAM 13-MAY-2017). In genomic DNA, the porcine TMC1 gene has 27 exons, wherein the 12th exon and its upper and lower 500bp sequences are shown in SEQ ID NO: 14, and its encoded protein fragment is shown in SEQ ID NO: 15.

[0126] 1. Conservation analysis of predetermined targets of TMC1 gene knockout and adjacent genome sequences

[0127] 18 newborn Congjiang pigs, including 10 females (named 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) and 8 males (named A, B, C, D , E, F, G, H).

[0128] Using the genomic DNA of 18 pigs as templates, PCR amplification was carried out using primer pairs (the target sequence of the primer pairs includes the 12th exon of porcine TMC1 gene), and then ...

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Abstract

The invention discloses a CRISPR / Cas9 system causing congenital deafness and application of the CRISPR / Cas9 system in preparation of model pig nuclear donor cells. The invention discloses a gRNA expression vector for a pig TMC1 gene. The gRNA expression vector expresses gRNA shown in SEQ ID NO: 25. The CRISPR / Cas9 system for editing the pig TMC1 gene comprises a Cas9 expression vector and the gRNAexpression vector disclosed by the invention, wherein the Cas9 expression vector is a plasmid with a complete sequence as shown in SEQ ID NO.2. Four gRNAs are designed for the pig TMC1 gene, efficient gRNAs are screened from the four gRNAs, and then preset targets are knocked out, so that the workload of later monoclonal cell identification and screening can be effectively reduced, and the gene editing efficiency can be detected directly through PCR product sequencing. When the Cas9 efficient expression vector modified by the system is used for gene editing, the editing efficiency is improvedby over 100 percent compared with that of the original vector.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a CRISPR / Cas9 system that causes congenital deafness and its application in preparing model pig nucleus donor cells, and more specifically relates to a CRISPR / Cas9 system and its application in the preparation of TMC1 gene mutations Application of porcine nuclear donor cells in cloning of congenital deafness. Background technique [0002] Congenital deafness refers to deafness caused by abnormalities in the process of pregnancy, childbirth or genetic factors, mostly sensorineural deafness. Congenital deafness can be divided into two categories: hereditary and non-hereditary. So far, 43 syndromic deafness genes and 99 non-syndromic deafness genes have been discovered. There are multiple genes that can cause autosomal dominant non-syndromic deafness and autosomal recessive non-syndromic deafness at the same time, and TMC1 is one of them. TMC1 was first reported in 2002 that its multip...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N15/62C12N15/12C12N5/10
CPCC12N15/113C12N15/85C12N5/0656C12N15/8509C12N9/22C07K14/47C12N2310/20C12N2800/107C12N2830/48C12N2830/50C07K2319/09C07K2319/60C12N2510/00A01K2227/108C07K2319/00A01K2267/0306
Inventor 牛冬汪滔马翔曾为俊王磊程锐陶裴裴赵泽英刘璐黄彩云
Owner NANJING KGENE GENETIC ENG CO LTD
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