Unlock instant, AI-driven research and patent intelligence for your innovation.

A kind of construction method of the vector that expresses multiple sgRNAs simultaneously

A construction method and expression vector technology, applied in the field of sgRNA, can solve problems such as limitations, and achieve the effect of ensuring openness

Active Publication Date: 2021-11-09
DONGHUA UNIV
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Comparing the existing Golden Gate-based sgRNA tandem expression methods, it can be found that the number of sgRNA tandem expression that these methods can achieve is limited by the number of sgRNA insertion plasmid subclones

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of construction method of the vector that expresses multiple sgRNAs simultaneously
  • A kind of construction method of the vector that expresses multiple sgRNAs simultaneously
  • A kind of construction method of the vector that expresses multiple sgRNAs simultaneously

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Construct a vector capable of simultaneously expressing 4 sgRNAs targeting 4 different genes

[0045] Step 1: Synthesize the insert fragment according to the sequence information in the table below, and construct four sgRNA expression vectors 42230-miR-505, 42230-miR-29a, 42230-miR-29c, 42230-SF2 according to the sgRNA insertion method in 42230 , targeting the genes miR-505, miR-29a, miR-29c and SF2, respectively. Sequencing verified the insertion of each sgRNA.

[0046] Table 1. Inserts in 42230

[0047]

[0048] Step 2: Insert the second sgRNA-miR-29a expression cassette at the EcoR I site of 42230-miR-505 to construct 42230-Multi-sgRNA-miR-505-miR-29a:

[0049] (1) According to the reaction system and procedure provided by the instruction manual of restriction endonuclease EcoR I of NEB Company, linearize 42230-miR-505, and the vector linearization result is as follows: figure 2 As shown in a, there is a band around 8500bp in the vector that has been linearize...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a method for constructing a vector for simultaneously expressing multiple sgRNAs, wherein n sgRNAs to be connected in series are respectively inserted into corresponding 42230 vectors; the sgRNAs are amplified by PCR amplification 2 The expression cassette is then seamlessly cloned in the first sgRNA 1 The EcoR I site on the expression vector implements the second sgRNA 2 Insertion of the expression cassette to obtain a vector that simultaneously expresses two sgRNAs; and then amplified by PCR to amplify the sgRNA 3 The expression cassette was then seamlessly cloned in the 42230‑sgRNA 1+2 The EcoR I site on the upper implementation of the third sgRNA 3 The insertion of the expression cassette results in a vector expressing three sgRNAs at the same time, and the operations are performed sequentially. The invention can realize the simultaneous knockout of gene family members at the same time, which solves the shortcomings of the existing methods.

Description

technical field [0001] The invention belongs to the sgRNA field of CRISPR / Cas9 system, and in particular relates to a method for constructing a vector for simultaneously expressing multiple sgRNAs. Background technique [0002] CRISPR (clustered regularly interspaced short palindromic repeats, clustered regulated interspaced short tandem repeats) originated from the immune system of bacteria and archaea. It is currently the most widely used gene editing tool and has been successfully applied to Escherichia coli, yeast, and mice , genome editing in humans and other species. [0003] According to the difference between CRISPR guide RNA and CRISPR-binding nuclease in the process of degrading target genes, CRISPR systems are divided into three categories. Among them, the second type of nuclease with Cas9 (CRISRP-associated) is the most used (PatrickD.Hsu, Eric S.Lander, and Feng Zhang.Development and Applications of CRISPR-Cas9for Genome Engineering.Cell 157,1263-1278(2014) )....

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/66C12N15/85
CPCC12N15/66C12N15/85C12N2800/107C12N2800/80C12N2810/10
Inventor 周宇荀李晓宁李文文王斯佳肖君华李凯
Owner DONGHUA UNIV