Circular RNA knockdown method and application thereof

A circular and protein technology, applied in the field of molecular biology, can solve the problems of non-specific targeting of parental RNA, low efficiency and off-target when circular RNA is knocked down, and achieve high-efficiency specific targeting, high design selectivity, and off-target less effect

Active Publication Date: 2020-12-25
GUANGZHOU GENESEED BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Based on the above problems, the purpose of the present invention is to solve the problems of low efficiency, off-target, and non-specific targeting of parental RNA when knocking down circular RNA. The method, which can effectively avoid off-targeting, has less interference with the homeostasis environment, makes the experimental results more authentic and reliable, has high efficiency of specific targeting knockdown, and does not non-specifically target parental genes and other beneficial effects

Method used

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  • Circular RNA knockdown method and application thereof
  • Circular RNA knockdown method and application thereof
  • Circular RNA knockdown method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 dCas13-HRSP12 vector construction

[0053] 1. PCR amplification of the target fragment

[0054] Using LwaCas13a-91902 plasmid, 293T cell cDNA and PCDH-3xFlag plasmid as templates, respectively amplified inactivated dLwaCas13a partial sequence D1 to 1459bp, inactive dLwaCas13a partial sequence D2 to 1735bp, inactive dLwaCas13a partial sequence and Linker and NES sequence D3 is 418bp, Linker and HRSP12 sequence D4 is 430bp, protein tag 3xFlag sequence D5 is 147bp, the specific sequence is as follows:

[0055] KD131-D1-UnF:GCTGGCTAGCGTTTAAACTTAAGCTTGCCACCATGAAAGTGACCAAGGTCGA

[0056] KD131-mut1-R:CCGTGAGCAATACTAGAGATGGCCTCATCGATATTGGCGAAGAAGTCCTCGATCT

[0057] Amplified size 1459bp;

[0058] KD131-mut1-F:CCATCTCTAGTATTGCTCACGGCATCGTGCATTTCAACCTGGAACTGGAAGGCAAG

[0059] KD131-mut2-R:GTTTGCAATATACAGGTCTTTTCTTCTCCTGCTTCAGTTTCTTCACTTTCT

[0060] The amplification size is 1735bp;

[0061] KD131-mut2-F:GAAGAAAGACCTGTATATTGCAAACTACATCGCTCACTTTAATTATATTCCTCAT

...

Embodiment 2

[0105] Embodiment 2 The design of gRNA and the construction of vector

[0106] Taking dLwaCas13a (KDHP-a) as an example, the design of the supporting gRNA and the construction of the vector include the following steps:

[0107] 1. Design of gRNA supporting dLwaCas13a

[0108] The gRNA supporting dLwaCas13a consists of DR plus target sequence, in the form of GATTTAGACTACCCCAAAAACGAAGGGGACTAAAACNNNNNNNNNNNNNNNNNNNNNNNNN, where N is the gRNA sequence.

[0109] The primer design software was used to retrieve the sequences at the circularization sites of circular RNAs circPVT1 and circHIPK3, and parameters such as GC content were calculated. The gRNA target sequences were designed as follows, and the corresponding vectors were named G11-G16 and G21-G27, respectively.

[0110] >gRNA targrt-circPVT1-1(11+11)

[0111] CTGGGCTTGAGGCCTGATCTTT

[0112] Among them, the brackets indicate the number of upstream bases at the cyclization site plus the number of downstream bases, that is, 1...

Embodiment 3

[0186] Example 3 Vector transfection and expression of KDHP protein

[0187] Using Lipofectamine 3000, dLwaCas13a (KDHP-a), dPspCas13b (KDHP-b), dRfxCas13d (KDHP-d) and dPspCas13b-truncated (983aa) (KDHP-bt) vectors were used to transfect 293T cells, and the samples were digested and centrifuged after 48 hours.

[0188] Wash 3 times with pre-cooled PBS, blot the residual liquid as much as possible, add 100 μL of cell lysate containing PMSF, lyse at low temperature for 20 minutes, and transfer the liquid to a 1.5 mL EP tube. Then add 5×Loading buffer (at a ratio of 2:1), boil at 100°C for 10 minutes and perform SDS-PAGE electrophoresis.

[0189] Using PVDF membrane, transfer membrane at 350mA for 80min. Then soak the membrane in blocking solution at 37°C for 1 h; take out the membrane and put it directly into the primary antibody (Flag antibody diluted 3000 times), overnight at 4°C; wash the membrane with PBST, 5min×4 times; transfer the membrane to the secondary antibody ( D...

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Abstract

The invention relates to the field of molecular biology, in particular to a circular RNA targeted knockdown method of and application thereof. According to the circular RNA knockdown method, dCas13 protein is used for fusion expression of HRSP12, dCas13 and matched gRNA are used for specific targeting of circular RNA, RNase P/MRP compounds are recruited through dCas13-HRSP12 fusion protein, specific targeting degradation of circular RNA is achieved, and the method has the beneficial effects that interference to in-vivo steady-state environment is little, experiment results are more real and reliable, and specific targeting knock-down efficiency is high.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a circular RNA knockdown method and application thereof. Background technique [0002] Circular RNAs (circular RNAs, circRNAs) are a class of circular RNA molecules mainly composed of more than one exon. They exist in large numbers in eukaryotic cells, and their expression has certain tissue and timing specificity, and is not easily detected by exonucleases. Degradation, more stable than linear RNA. Circular RNA can act as a miRNA sponge to regulate host transcriptional activity, directly translate proteins, affect gene regulation and expression, and play an important role in brain development, Parkinson's disease, Alzheimer's disease and tumorigenesis, and is expected to become a new type of clinical disease. diagnostic markers. [0003] Most circRNAs are produced by back-splicing exons of protein-coding genes (host genes). Because they are back-spliced ​​into a ci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N9/22C12N15/867C12N7/01
CPCC07K14/47C07K2319/43C12N7/00C12N9/22C12N15/113C12N15/86C12N2740/15021C12N2740/15043
Inventor 刘明李自强王超刘景磊蔡秋杰
Owner GUANGZHOU GENESEED BIOTECH
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