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Fusion protein based on CjCas9 and VPR core structure domains, corresponding DNA targeting activation system and application of system

A DNA targeting and fusion protein technology, applied in recombinant DNA technology, DNA/RNA fragments, stably introducing foreign DNA into chromosomes, etc., can solve the problems of low targeting, too large system size, and low activation efficiency

Active Publication Date: 2019-10-22
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to combine the currently smallest CRISPR / Cas9 system protein CjCas9 with the transcription activator VPR to solve the problems of low targeting, low activation efficiency, and too large system volume for in vivo application in existing gene activation methods. Provide an activation system with small size, strong activation effect and high specificity, which can realize efficient gene activation in vivo and in vitro

Method used

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  • Fusion protein based on CjCas9 and VPR core structure domains, corresponding DNA targeting activation system and application of system
  • Fusion protein based on CjCas9 and VPR core structure domains, corresponding DNA targeting activation system and application of system
  • Fusion protein based on CjCas9 and VPR core structure domains, corresponding DNA targeting activation system and application of system

Examples

Experimental program
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Effect test

Embodiment 1

[0074] Construct a vector expressing dCjCas9 protein: Digest the purchased pRGEN-CMV-CjCas9 (89752) plasmid with Nco1 to obtain a 3383bp fragment as a vector, and use pRGEN-CMV-CjCas9 as a template to perform PCR amplification. The PCR1 primer sequence is as follows:

[0075] F1: GTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC; (SEQ ID NO: 11)

[0076] R1: AGCGAAGGCCAGGATTCTGGCCATGATTCGGATCCCAAGCTTG; (SEQ ID NO: 12)

[0077] The PCR2 primer sequences are as follows:

[0078] F2: GCCAGAATCCTGGCCTTCGCTATCGGCATCAGCAGCATCG; (SEQ ID NO: 13)

[0079] R2: ACCGGCTGTAGGGGTAGATGGCGTCGATTTCCAGCATCTTCT; (SEQ ID NO: 14)

[0080] The PCR3 primer sequences are as follows:

[0081] F3: AAGATGCTGGAAATCGACGCCATCTACCCCTACAGCCGGT; (SEQ ID NO: 15)

[0082] R3: AGCACCTTCAGGGCGAAGTCCATTGTGTAGATGGGCACGGCGTA; (SEQ ID NO: 16)

[0083] The above four fragments were ligated using Gibson Assembly reagent to obtain the pRGEN-CMV-dCjCas9 plasmid.

[0084] Enzyme digestion system 50ul, including:

[0085] Ve...

Embodiment 2

[0094] Construction of a vector expressing VPR protein: using pBlu2KSP as a vector, digesting the vector fragment with kpn1 and spe1, and using pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS (60904) as a template, perform PCR amplification to obtain vp64 , PCR1 primer sequence is as follows:

[0095] F1: CTCACTATAGGGCGAATTGGGTACCGATGCTTTAGACGATTTTGA; (SEQ ID NO: 17)

[0096] R1: CCTCCTCCTCCGCTTCCTCCTAGCATATCTAGATCAAAGT; (SEQ ID NO: 18)

[0097] Using MS2-P65-HSF1_GFP (61423) as a template, perform PCR amplification to obtain p65, and the PCR2 primer sequence is as follows:

[0098] F2: ACTTTGATCTAGATATGCTAGGAGGAAGCGGAGGAGGAGG; (SEQ ID NO: 19)

[0099] R2: AGGTCGCTGCCGCTGCCAGACCCACTAGAGGAAATCTGT; (SEQ ID NO: 20)

[0100] Using the synthesized rta sequence as a template, perform PCR amplification to obtain rta, and the PCR3 primer sequence is as follows:

[0101] F3: ACAGATTTCCTCTAGTGGGTCTGGCAGCGGCAGCGACCT; (SEQ ID NO: 21)

[0102] R3: CGGTGGCGGCCGCTCTAGAAAACAGAGATGTGTCGAAGA; (SEQ I...

Embodiment 3

[0106] Construct the vector expressing the fusion protein VPR-dCjCas9: use pRGEN-CMV-dCjCas9 as the vector, digest the vector fragment with Bamh1, and use pBlu-VPR as the template to perform PCR amplification to obtain VPR. The PCR1 primer sequence is as follows:

[0107] F1: ATAGGGAGACCCAAGCTTGGGCCACCATGGATGCTTTAGACGATTTTGA; (SEQ ID NO: 23)

[0108] R1: GGGGAGCCGCTGCCCAGGCTAAACAGAGATGTGTCGAAGA; (SEQ ID NO: 24)

[0109] Using pCAG-dCas9-24xGCN4_v4-NLS-P2A-BFP as a template, perform PCR amplification to obtain the linker sequence, and the PCR2 primer sequence is as follows:

[0110] F1: TCTTCGACACATCTCTGTTTAGCCTGGGCAGCGGCTCCC; (SEQ ID NO: 25)

[0111] R1: AGGATTCTGGCCATGATTCGTCCTCCAGAACCTCCACCTC; (SEQ ID NO: 26)

[0112] Utilize Gibson Assembly reagent to connect above-mentioned 3 fragments to obtain pRGEN-CMV-VPR-Linker-dCjCas9 plasmid (such as figure 1 shown).

[0113] Specific steps such as enzyme digestion system, PCR amplification, and Gibson Assembly reaction system a...

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Abstract

The invention discloses a fusion protein based on CjCas9 and VPR core structure domains, a corresponding DNA targeting activation system and application of the system. The fusion protein comprises twoheterologous polypeptide structure domains, and one polypeptide structure domain comprises VPR protein with the transcription activation activity; the other polypeptide structure domain comprises theCjCas9 protein, the CjCas9 protein is in a dCjCas9 subtype or a mini-dCjCas9 subtype or a CjCas9 wild type, and the dCjCas9 subtype contains D8A and H559A single-locus amino acid mutation; the mini-dCjCas9 subtype contains D8A single-locus amino acid mutation and deficiency of most HNH structure domains. The DNA targeting activation system comprises the fusion protein and one or more kinds of guide RNA, and the guide RNA comprises a sequence with the length of 14-22 bp and a framework sequence with the length of 80 bp, wherein the sequences are designed for a target gene promoter area. The DNA targeting activation system is applied to targeting activation of a gene. The DNA targeting activation system has the advantages of being small in size, capable of achieving high specificity, a highactivation effect and easy synthesis, low in cost and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a fusion protein based on CjCas9 and its mutants and a VPR core domain, a corresponding DNA targeting activation system and applications thereof. Background technique [0002] The CRISPR / Cas9 system is currently a widely used gene editing system, which is transformed from the adaptive immune mechanism of bacteria and archaea to degrade foreign DNA. The CRISPR / Cas9 system mainly consists of two components: Cas9 protein and guide RNA. After the combination of Cas9 protein and guide RNA, it will cut the double strand of DNA at a specific site under the guidance of guide RNA, resulting in a break, which is much simpler than the previous gene editing method. In recent years, the CRISPR / Cas9 system has also been gradually applied in the field of gene regulation. Using the CRISPR / Cas9 system that has lost its cleavage activity and combined with transcriptional activators or repressors can a...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/113C12N15/85C12N15/90
CPCC07K14/4702C07K2319/00C12N9/22C12N15/113C12N15/85C12N15/902C12N2310/10C12N2310/20
Inventor 荣知立林瑛张鑫彭欣
Owner SOUTHERN MEDICAL UNIVERSITY
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