Method for screening target gene using CRISPRI system and uses thereof
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.
1 . 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.
2 . The method for constructing the shortened sgRNA random library of claim 1 , wherein the shortened TRS consists of 9 nucleotides from the 5′-terminal.
3 . The method for constructing the shortened sgRNA random library of claim 1 , wherein the complementary binding comprises complete complementary binding or one or more mismatch bindings to the target DNA.
4 . The method for constructing the shortened sgRNA random library of claim 3 , wherein the target DNA comprises a nucleotide of a complementary sequence to the TRS or sgRNA and a protospacer-adjacent motif (PAM).
5 . The method for constructing the shortened sgRNA random library of claim 1 , wherein the shortened sgRNA comprises crRNA including a shortened TRS and tracrRNA.
6 . 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.
7 . The method for screening the target gene based on the CRISPRi system of claim 6 , further comprising:
additionally screening a target gene based on a duplicated gene in a shortened sgRNA random library comprising shortened TRSs having the same length or a duplicated gene between shortened sgRNA random libraries comprising shortened TRSs having the various lengths, among the target genes screened in the step (e).
8 . The method for screening the target gene based on the CRISPRi system of claim 6 , wherein the dCas protein is a dead CRISPR/Cas enzyme selected from the group consisting of dead Cas9, dead Cas12a, dead Cas12b, and dead Cas12c.
9 . The method for screening the target gene based on the CRISPRi system of claim 8 , wherein the dCas protein is dCas9 comprising mutations in HINH and RuvC domains.
10 . The method for screening the target gene based on the CRISPRi system of claim 9 , wherein the dCas9 protein is derived from a bacterial species selected from the group consisting of Streptococcus pyogenes, Francisella novicida, Streptococcus thermophilus, Legionella pneumophila, Listeria innocua and Streptococcus mutans.
11 . The method for screening the target gene based on the CRISPRi system of claim 10 , wherein the dCas9 protein comprises at least one mutation selected from the group consisting of D10A, H840A and N863A in Streptococcus pyogenes Cas9.
12 . The method for screening the target gene based on the CRISPRi system of claim 6 , wherein the target gene is involved in xylose catabolism.
13 . 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.