Method for screening target gene using CRISPRI system and uses thereof
AssigneeChungang University Industry Academic Cooperation Foundation
InventorsSang Jun Lee, Hyun Ju Kim, Song Hee Jeong
Life of this patent
Estimating the term…
Abstract
The present invention relates to a method for screening a target gene using a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system and uses thereof. According to the present disclosure, it is expected that the method may be used in wide fields as a tool for genome-scale gene function profiling and studying organisms poorly developed in genetic technology, including pathogens or industrially useful strains or cell lines, and as a platform for electronic regulatory network engineering of genes.
Claims13
A method for constructing a shortened sgRNA random library comprising the following steps:
- (a) synthesizing shortened crRNA that complementarily binds to target DNA, wherein the shortened crRNA comprises a shortened target recognition sequence (TRS) consisting of the length selected from the group consisting of 7 to 19 nucleotides from a 5′-terminal;
- and
- (b) generating a shortened sgRNA random library including the shortened crRNA by repeating the step of synthesizing the shortened crRNA one or more times.
A method for screening a target gene based on a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system comprising the steps of:
- (a) synthesizing shortened crRNA that complementarily binds to target DNA, wherein the shortened crRNA comprises a shortened target recognition sequence (TRS) consisting of the length selected from the group consisting of 7 to 19 nucleotides from a 5′-terminal;
- (b) generating a shortened sgRNA random library including the shortened crRNA by repeating the step of synthesizing the shortened crRNA one or more times;
- (c) introducing a the shortened sgRNA random library prepared in the step (b) prepared into plurality of cells in which a nuclease-deactivated Cas (dCas) protein is overexpressed, thereby generating a plurality of test transformants;
- (d) selecting a subject exhibiting a modified phenotype among the transformants, compared to a control group;
- and
- (e) confirming a sequence of a shortened TRS of the shortened sgRNA introduced into the selected subject, thereby screening a target gene exhibiting high activity.
A method for mass-producing a target product of interest comprising the following steps:
- (a) synthesizing shortened crRNA that complementarily binds to target DNA, wherein the shortened crRNA comprises a shortened target recognition sequence (TRS) consisting of the length selected from the group consisting of 7 to 19 nucleotides from a 5′-terminal;
- (b) generating a shortened sgRNA random library including the shortened crRNA by repeating the step of synthesizing the shortened crRNA one or more times;
- (c) introducing the shortened sgRNA random library prepared in the step (b) into plurality of cells in which a nuclease-deactivated Cas (dCas) protein is overexpressed, thereby generating a plurality of test transformants;
- (d) selecting a subject exhibiting a modified phenotype among the transformants, compared to a control group;
- (e) confirming a sequence of a shortened TRS of the shortened sgRNA introduced into the selected subject, thereby screening a target gene exhibiting high activity;
- (f) deleting two or more target genes screened in the step (e) in a host cell to produce a target product of interest;
- and
- (g) incubating the host cell to obtain a mass-produced target product of interest.
Dependencies are inferred from the claim text.