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Composite multi-connected gRNA and RNAi expression cassette for vector expression

A kind of framework and carrier technology, applied in the field of expression framework of composite multiplex gRNA and RNAi used for vector expression

Active Publication Date: 2015-10-28
许中伟 +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] In order to overcome the shortcomings of the above-mentioned existing single or multiple gRNA expression frameworks and achieve compound, multiple, and convenient gene editing functions, the present invention provides a technical solution, which is a new multi-gRNA and RNAi expression framework, which is characterized by Between a promoter and a terminator in a CRISPR / Cas9 expression vector, there is a "composite, multiple gene editing expression framework" with one or more gRNAs and one or more RNAi

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  • Composite multi-connected gRNA and RNAi expression cassette for vector expression
  • Composite multi-connected gRNA and RNAi expression cassette for vector expression
  • Composite multi-connected gRNA and RNAi expression cassette for vector expression

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Embodiment 1

[0069] Example 1. Construction of Expression Framework

[0070] Segmented oligonucleotides are first synthesized, and then the specific fragments are inserted into the vector in sequence, and the expression framework is constructed by step-by-step ligation. For details, please refer to the description below.

[0071] Such as figure 1 The construction of expression framework 1 shown in a, in this embodiment, the spacer sequence is the flanking sequence of pre-miRNA in the pri-miRNA sequence or similar sequence.

[0072] 1. Construction of promoter-gRNA1-spacer1-RNAi1-spacer2-gRNA2-terminator expression framework

[0073]1) Use the rapid mutation method to insert XhoI and HindIII restriction sites into the p458 (pSpCas9(BB)-2A-GFP) vector, and remove the poly T sequence to construct a p458M vector. The specific positions are as follows: figure 2 shown.

[0074] 2) Synthesizing oligonucleotides, performing denaturation, annealing and ligation in sequence, and loading gRNA1 in...

Embodiment 2

[0098] Example 2 Expression Framework 1 Result Evaluation

[0099] Take miR-HBV with the length of the spacer as a bridge to connect two HBV-specific gRNAs, p458M-gRNA1-XhoI-spacer1-miR-HBV-spacer2-BamHI-gRNA2 terminator as an example.

[0100] 3-H51-2 means: spacer sequence 1 and spacer sequence 2 are the flanking sequences of pri-miR-31, both of which are 51nt in length; 3-H38-2 means: the length of spacer sequence 1 is both 38nt, and the length of spacer sequence 2 is 3-H30-2 means: the length of spacer sequence 1 is 30nt, the length of spacer sequence 2 is 31nt; 3-H22-2 means: the length of spacer sequence 1 is 22nt, and the length of spacer sequence 2 is 21nt .

[0101] The MiR-HBV sequence is a microRNA sequence that can specifically act on HBV after being modified on the basis of the HBV sequence.

[0102] Among them, the specific design principles and sequences are as follows: image 3 shown.

[0103] image 3 It is a diagram of the principle and sequence of the H...

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Abstract

The invention provides a composite multi-connected gRNA and RNAi expression cassette for vector expression. The composite multi-connected gRNA and RNAi expression cassette can be applied to a eukaryotic expression vector system. The composite multi-connected expression cassette comprises a promoter for promoting transcription, one or more gRNA expression cassette bodies, one or more RNAi expression cassette bodies and a terminator for terminating transcription; the expression cassette has the function of modifying target genes at the DNA level and the mRNA level, and the gRNA / CRISPER gene modifying expression cassette bodies are connected with the RNAi expression cassette bodies through intervening sequences in an alternative series connection mode. The composite multi-connected gRNA and RNAi expression cassette for vector expression solves the problem that in an existing expression structure, one promoter can only be connected with one gRNA and one transcription terminator.

Description

technical field [0001] The present invention relates to an expression framework containing compound and multiple gRNA and RNAi expressed in eukaryotic cells and its construction. Background technique [0002] The CRISPR / Cas system is a new tool for targeted editing of genomic DNA developed in recent years. This system was originally a defense system in bacteria against foreign DNA such as viruses. There are a series of clustered DNA sequences in some bacterial genomes, called "Clustered Regularly Interspaced Short Palindromic Repeats" (CRISPR). The spacers between these repeats were found to be identical to the DNA sequences of many phages capable of invading bacteria. Moreover, after these sequences are transcribed into RNA, they can form a complex with a type of protein called Cas (CRISPR associated) produced by the host bacterium to guide the Cas protein, so this piece of RNA is also called guide RNA (guide RNA, gRNA). When the complex detects that the invading DNA an...

Claims

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Application Information

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IPC IPC(8): C12N15/85A61K48/00A61P31/20A61P31/18A61P35/00
Inventor 许中伟鲁凤民王杰
Owner 许中伟
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