Guide RNA and genetic modification methods for the host microorganism Escherichia coli to induce target gene expression using the CRISPR / CAS system.

Abstract

OCR06WT The current invention involves guide RNA (gRNA) for use in modification. Genetic precursors of host microorganisms and methods for genetic modification of host microorganism species. Escherichiacoli was used to induce the expression of target genes using the CRISPR / CASS system. Such genetic modification involves introducing a target gene into a specific intergene region. of the host microorganisms using a CRISPR / Case system in which the CRISPR / Case system is comprised of (5) A plasmid vector guide RNA (gRNA plasmid) containing specific guide RNA (gRNA). To modify the desired position on the specific intergene region (i) plasmid vector redcas-CM. (REDCas-Cmplasmid) and Gii) prototypes for CAMP repair consisting of nucleotide sequences. Target gene removal and degradation of plasmid welter-guided RNA and plasmid vector redcast. CM is released from genetically modified microorganisms containing a guide RNA nucleotide sequence. Choose one or more nucleotide sequences from 1-38 (SEQIDNO:1-38). Combined

Claims

OCR10KL(15 / 05 / 2569) 1. Guide RNA (gRNA) for use in genetic modification of a host microorganism in which the guide RNA contains one or more 5'-3' nucleotide sequences from sequences 1-38 (SEQIDNO:1-38).

2. Guide RNA for use in genetic modification of a host microorganism according to claim 1 in which the appropriate host microorganism is Escherichia acolí.

3. Guide RNA for use in genetic modification of a host microorganism according to claim 2. Where the most suitable host microorganism Escherichiacoli can be selected from EscherichiacoliC, EscherichiacoliMYR428, EscherichiacoliMYR1674, modified EscherichiacoliC (EscherichiacoliC derivatives), modified EscherichiacoliMYR428 (EscherichiacoliMYR428 derivatives), and modified EscherichiacoliMYR1674 (EscherichiacoliMYR1674 derivatives).

4. Guide RNA for use in genetic modification of the host microorganism according to claim 1, prepared by the CRISPR / CAS system. 5.Guide RNA for use in genetic modification of host microorganisms under Patent 4, whereby the appropriate CRISPR / Cas system can be selected from CRISPR / Cas9, CRISPR / Cas12, CRISPR / Cas13, and CRISPR / dCas9 systems.

6. Guide RNA for use in genetic modification of host microorganisms under Patent 5, whereby the most suitable CRISPR / Cas system is CRISPR / Cas9.

7. Guide RNA for use in genetic modification of host microorganisms under Patent 1, whereby genetic modification can be selected from substitution, insertion, addition, deletion, detection, interference, and repression. 8.The CRISPR / CAS system for use in genetic modification of host microorganisms consists of (i) a gRNA plasmid vector containing a guide RNA according to Patent 1 that is specific to the intergene region, (ii) a REDCas-Cmplasmid vector, and (iii) a repair template containing the nucleotide sequence of the target gene.

9. The CRISPR / CAS system for use in genetic modification of host microorganisms according to Patent 8, in which the host microorganism The most suitable host is Escherichiacoli10. The CRISPR / CAS system for use in genetic modification of the host microorganism under Patent 9, in which the most suitable host microorganism Escherichiacoli can be selected from EscherichiacoliC, EscherichiacoliMYR428, EscherichiacoliMYR1674, modified EscherichiacoliC (EscherichiacoliC derivatives), modified EscherichiacoliMYR428 (EscherichiacoliMYR428 derivatives), and modified EscherichiacoliMYR1674 (EscherichiacoliMYR1674 derivatives).11.The CRISPR / CASS system for use in genetic modification of host microorganisms under Patent 8, in which the plasmid vector guide RNA is further composed of: a. a promoter and terminator specific to the guide RNA, b. a DNA replication site, and c. an antibiotic selection gene, where the guide RNA is placed between the promoter and terminator with the DNA replication site and the antibiotic selection gene is placed at the remaining locations.

12. The CRISPR / CASS system for use in genetic modification of host microorganisms. The host microbial genetic material under claim 8, in which the REDCas-Cmplasmid plasmid vector comprises: a. a first gene set containing genes inducing the expression of a Cas enzyme specific to the nucleotide sequence of the guide RNA; b. a second gene set for the Lambda-red system containing genes inducing the expression of the Lambda-red system, which consists of the Gam, beta, and exo proteins; and c. promoters and terminators that control the expression of the Cas, Gam, beta, and exo genes.

12. The origin of DNA replication and the selection gene for antibiotic use, where the promoter is placed anteriorly to the first and second gene sets, with the terminator placed posteriorly to that gene set, and where the origin of DNA replication and the selection gene for antibiotic use are placed at the remaining positions.

13. The CRISPR / CAS system for use in genetic modification of host microorganisms under claim 8, where the repair template has a nucleotide sequence size in the range of 100-10,000 base pairs.

14. The CRISPR / CAS system for use in genetic modification of host microorganisms under claim 8, where the repair template has a target gene selected from a group of genes that induce the expression of fluorescent proteins, a group of genes that induce transcription, and a group of genes that induce the expression of proteins or enzymes involved in product synthesis.

15. The method of genetically modifying host microorganisms to express the target gene consists of the following steps: a.

16. Method for genetically modifying the host microorganism to achieve target gene expression according to Patent 15, where the optimal host microorganism is Escherichiacoli.

17. Method for genetically modifying the host microorganism to achieve target gene expression according to Patent 16, where the optimal host microorganism is Escherichiacoli from Escherichiacoli C, Escherichiacoli MYR428, Escherichiacoli MYR1674, modified Escherichiacoli Cn (Escherichiacoli C derivatives), modified Escherichiacoli MYR428 (Escherichiacoli MYR428 derivatives), and modified Escherichiacoli MYR1674 (Escherichiacoli MYR1674 derivatives).

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