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DNA system for selectively killing cells by inducing multi-site cutting genome by utilizing RNA interference mechanism

An RNA interference and genomic technology, applied in the field of DNA systems, can solve the problems of complex redundancy and correlation, side effects, and no obvious regulatory effect.

Pending Publication Date: 2022-01-07
NANJING RAYGEN HEALTH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since each miRNA can regulate the expression of hundreds of target genes on average, and each target gene is often under the regulation of multiple miRNAs, it presents very complex redundancy and correlation.
Therefore, by reducing or delivering a single or a small number of miRNAs to regulate the expression level of disease-causing genes, on the one hand, there may be no obvious regulatory effect, and on the other hand, it may bring serious side effects

Method used

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  • DNA system for selectively killing cells by inducing multi-site cutting genome by utilizing RNA interference mechanism
  • DNA system for selectively killing cells by inducing multi-site cutting genome by utilizing RNA interference mechanism
  • DNA system for selectively killing cells by inducing multi-site cutting genome by utilizing RNA interference mechanism

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Materials and methods

[0071] Construction of plasmids and vectors

[0072] Cas9 was ligated into a piggyBac vector containing a hygromycin resistance gene driven by a PGK promoter driven by a CAGGS promoter. The pre-sgRNA (sgRNA sequence containing RNA sequences completely complementary to the target miRNA or siRNA at both ends, referred to as miRT-sgRNA-miRT) was connected to the bleomycin (zeocin) resistance gene driven by the PGK promoter. In the piggyBac vector, the pre-sgRNA is also driven by the CAGGS promoter. In order to prepare the miRT-sgRNA-miRT construct sensitive to target miRNA or siRNA, use Fastpfu polymerase (Transgene Company), with the plasmid containing sgRNA as a template (the sequence of the sgRNA is complementary to the partial sequence of the Alu element), with Primers corresponding to miRT were used for PCR. The PCR product was incubated with EcoRI-HF (20U, NEB Company) and BamHI-HF (20U, NEB Company) at 37°C for 2h, purified with a spin c...

Embodiment 2

[0083] To test the killing effect of MICR-CUT system on Hela cells.

[0084] In this study, the Alu element with a high repetitive sequence in the Hela cell genome was first selected, and two sgRNAs targeting the Alu element were designed. -OFFinder software forecast). In this study, these two sgRNAs were constructed as pre-sgRNAs (referred to as 294T-Alu1, 294T-Alu2) that can be activated by miR-294. See the experimental results figure 2 , indicating that Cas9 with nuclease activity and pre-sgRNA expression vectors were transfected into Hela cells, and both pre-sgRNAs could cause about 50% reduction in the number of Hela cells under the condition of miR-294 transfection. The experimental results preliminarily indicate that the MICR-CUT DNA system can be used to specifically kill cells by targeting and cutting repetitive sequences.

Embodiment 3

[0086] Using the pre-sgRNA tandem MICR-CUT system to specifically kill Hela cells.

[0087] In order to enhance the killing effect of the MICR-CUT system, this study chose to connect two pre-sgRNAs targeting different sites in series to form a single pre-sgRNA, so that when the miRNA activates the pre-sgRNA, it can simultaneously produce two different sgRNAs, respectively targeting different genomic repeat sites. In this study, miR-294 and miR-21 targeted activation pre-sgRNAs (referred to as 21T-Alu1-Alu2; 294T-Alu1-Alu2 for short) were constructed.

[0088] See the experimental results image 3 , it can be seen that: after transfection of Cas9 and 21T-Alu1-Alu2 expression plasmids, the number of Hela cells was significantly reduced; while transfection of Cas9 and 294T-Alu1-Alu2 had no significant effect on the growth of Hela cells, only when exogenous transfection of miR -294 can significantly reduce the number of Hela cells. The experimental results further prove that th...

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Abstract

The invention provides a DNA system for selectively killing cells by inducing a multi-site cutting genome by using an RNA interference mechanism. A cell killing system comprises the following parts: 1, a plasmid for expressing a nuclease CRISPR-Cas9 protein; and 2, a plasmid for expressing a precursor single-stranded-guided RNA (pre-sgRNA), wherein a precursor synthesized guide RNA contains a sequence which is completely complementary with a target miRNA or siRNA and a sequence of a synthesized guide RNA (sgRNA); and the killing system is used for inducing a CRISPR / Cas9 cutting genome by utilizing miRNA or siRNA specifically expressed by the cells, so that targeted killing of the cells is realized.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a DNA system for selectively killing cells by using an RNA interference mechanism to induce multi-site cutting of genomes. Background technique [0002] Mechanism of RNA interference [0003] RNA interference (RNA interference, RNAi) refers to the phenomenon of efficient and specific degradation of homologous mRNA induced by single-stranded or double-stranded RNA (double-stranded RNA, dsRNA). RNAi has the following characteristics: 1) RNAi is a gene silencing mechanism at the post-transcriptional level; 2) RNAi has high specificity, and only degrades the mRNA of a single endogenous gene corresponding to its sequence; 3) RNAi inhibits gene expression with high The efficiency, the phenotype can reach the level of the deletion mutant phenotype, and a relatively small amount of dsRNA molecules (the amount is far less than the amount of endogenous mRNA) can completely inhibit the ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/113C12N9/22
CPCC12N15/85C12N15/113C12N9/22C12N2310/20C12N2800/107
Inventor 汪阳明胡鲁峰
Owner NANJING RAYGEN HEALTH CO LTD