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Preparation method and application of Cas9-RNAi RNP with efficient homologous directional repair activity

An active and efficient technology, applied in the field of recombinant proteins, can solve problems such as ectopic rearrangement of chromatin, inability to repair cells in time, and cancerous cell death, etc., and achieve the effects of efficient repair, cost reduction and efficiency improvement

Pending Publication Date: 2022-06-28
BRAVOVAX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whole-cell or continuous inhibition of the NHEJ pathway will cause cells to fail to repair other DNA damage in time, resulting in chromatin heterotopic rearrangement, cell cancer death and other consequences

Method used

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  • Preparation method and application of Cas9-RNAi RNP with efficient homologous directional repair activity
  • Preparation method and application of Cas9-RNAi RNP with efficient homologous directional repair activity
  • Preparation method and application of Cas9-RNAi RNP with efficient homologous directional repair activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction of Cas9-RNAi RNP expression plasmid

[0046] 1. The Ptac-cas9-T7sgRNA plasmid stored in our laboratory is the backbone of the vector, which is digested with the restriction enzyme SalI. The SpCas9 gene sequence already exists on the plasmid, and the sgRNA-shRNA sequence only needs to be connected after the restriction site of SalI.

[0047] 2. Design sgRNA-shRNA sequences targeting BFP gene and interfering Ligase 4 gene with a total length of 210nt. Five pairs of primers were synthesized and the sgRNA-shRNA fragments were constructed by annealing the homology arms, and then PCR amplification was carried out. When replacing the sgRNA sequence and shRNA sequence, only 2 pairs of primers need to be redesigned, and the other 3 pairs of primers remain unchanged. The mixed primer in overlapping PCR is to mix and dilute other primers except the first and last primers by 10 times. The cycle number of overlapping PCR is about 10. The product is used as a...

Embodiment 2

[0073] Example 2 Expression purification and in vitro activity test of Cas9-RNAi RNP

[0074] like figure 1 As shown, the Cas9-RNAi RNP expression plasmid constructed in Example 1 was transformed into E. coli expression strain HT115 DE3 competent cells, and the target expression strains were obtained by screening with ampicillin and tetracycline double antibodies. Introduce this strain into the liquid medium containing double antibody, cultivate overnight in a shaker at 37°C 220rpm, then transfer to 1L liquid medium containing corresponding antibiotics at 1:100, and continue to culture at 37°C 220rpm to OD600 =0.6~0.8. After adding IPTG with a final concentration of 1 mM, it was transferred to a shaker at 18°C ​​and 220 rpm for induction for 16-18 hours. After induction, the cells were collected and disrupted, and a large number of Cas9-RNAi RNPs were rapidly prepared by one-step Ni-bead affinity purification and analyzed by SDS-PAGE electrophoresis. The purification result...

Embodiment 3

[0076] Example 3 Detection of the integrity of sgRNA-shRNA in Cas9-RNAi RNP

[0077] In order to detect whether the protein obtained in Example 2 contains complete sgRNA-shRNA, the purified Cas9-RNAi RNP was treated with proteinase K, and the results of polyacrylamide gel nucleic acid electrophoresis were as follows: image 3 shown. The theoretical size of sgRNA-shRNA is about 210nt, and the electrophoresis results are consistent with expectations.

[0078] The results of this example show that the constructed Cas9-RNAi RNP expression plasmid can be correctly expressed when transformed into E. coli HT115(DE3), and the self-assembled SpCas9 RNP containing sgRNA-shRNA structure in E. coli can be obtained by Ni-bead affinity purification in one step.

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Abstract

The invention discloses a preparation method of Cas9-RNAi RNP with efficient homologous directional repair activity, which comprises the following steps: transiently inhibiting key protein Ligase 4 of an NHEJ pathway, constructing a similar structure of shRNA by prolonging the 3'end of gRNA of Cas9RNP, and expressing Cas9RNP by using escherichia coli cells in a one-step method to obtain Cas-RNAi RNP carrying specific sgRNA-shRNA. According to the Cas-RNAi RNP disclosed by the invention, the function combination of gene editing and RNA interference is realized, and the shRNA base sequence aiming at the Ligase 4 gene is designed so as to temporarily inhibit the NHEJ pathway, so that efficient HDR repair is realized.

Description

technical field [0001] The invention relates to a preparation method and application of a Cas9-RNAi RNP with efficient homology directional repair activity, and belongs to the field of recombinant proteins. Background technique [0002] The CRISPR system is derived from the acquired immune system of bacteria and archaea, which can target and cut the foreign genetic material that invades again under the guidance of crRNA. Inspired by this process, the researchers developed a genome editing technique called CRISPR / Cas9. The main components of the system are Cas9 protein and single-stranded guide RNA (single guide RNA, sgRNA). Cas9 protein reaches the target gene under the guidance of sgRNA and the base pairing of specific gene sequence, and cuts DNA through HNH and RuvC domains Causes a double strand break (DSB). In mammalian cells, the two major pathways for the repair of such DSBs are non-homologous end joining (NHEJ), where insertion or deletion mutations are predominant,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/55C12N15/113C12N9/22C12N15/88
CPCC12N15/70C12N9/22C12N15/113C12N15/1137C12N15/88C12N2310/20C12N2310/14C12N2310/531
Inventor 刘奕孙文丽吴克殷文浩乔洁
Owner BRAVOVAX
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