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Method for simultaneously realizing gene editing and transcriptional regulation by using I-type CRISPR-Cas system

A technology of transcriptional regulation and gene editing, applied in the biological field, can solve problems such as inability to complete gene editing and transcriptional regulation at the same time

Pending Publication Date: 2022-06-24
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, in the past, gene editing and transcriptional regulation could not be accomplished simultaneously with one CRISPR-Cas system

Method used

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  • Method for simultaneously realizing gene editing and transcriptional regulation by using I-type CRISPR-Cas system
  • Method for simultaneously realizing gene editing and transcriptional regulation by using I-type CRISPR-Cas system
  • Method for simultaneously realizing gene editing and transcriptional regulation by using I-type CRISPR-Cas system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] The crtB gene of S. spainata, as shown in SEQ ID NO: 1 of the sequence listing, encodes a phytoene synthase (HAH_2563). Knockout of this gene will block the expression of carotenoids and turn the cells from red to white.

[0075] Both the Cascade effector and the Cas3 endonuclease are endogenous to H. spainii.

[0076]In this example, for the crtB gene of S. spain, using its own type I-B CRISPR-Cas system, by using gRNAs with spacers of different lengths, different degrees of transcriptional inhibition of the crtB gene can be achieved without cleaving it.

[0077] 1. Construction of plasmids for expressing gRNAs with spacers of different lengths

[0078] 1. Preparation of seven PphaR-R-spacer-R fragments

[0079] Our laboratory has constructed a gRNA expression plasmid pWL502-PphaR-R-C1-R (namely plasmid pGK) targeting the crtB gene of the DLCR strain of S. spainata in the early stage, which contains the PphaR-R-C1-R fragment. The PphaR-R-C1-R fragment is shown in SE...

Embodiment 2

[0131] The cdc6E and crtB genes were simultaneously edited and transcriptionally regulated by the I-B CRISPR-Cas system of the HH DLCR strain of S. The crtB gene is shown in SEQ ID NO: 1 of the sequence listing. The cdc6E gene is shown in SEQ ID NO: 10 of the Sequence Listing.

[0132] 1. Construction of editing and transcriptional regulation plasmid pGKBE-DonorE-SpacerBE

[0133] 1. Preparation of DonorE-SpacerBE fragments

[0134] There is a knockout plasmid pGKBE targeting the cdc6E gene and crtB gene of the DLCR strain of S. spainii, expressing a gRNA with two spacers and containing the Donor sequence required for knocking out the crtB gene and the Donor required for knocking out the cdc6E gene sequence. The two spacers are the spacers targeting the crtB gene (S B ) and a spacer (S) targeting the cdc6E gene E ). In the gRNA expressed by plasmid pGKBE, S B The length is 36nt (positions 269-304 of the crtB gene shown in targeting sequence 1, PAM sequence is TTC), S E...

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Abstract

The invention discloses a method for simultaneously realizing gene editing and transcriptional regulation by using an I-type CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated proteins) system. The method provided by the invention comprises the following steps: carrying out transcriptional regulation on a gene A by utilizing an I-type CRISPR-Cas system A, and carrying out gene editing on a gene B by utilizing an I-type CRISPR-Cas system B; a target gene of the I-type CRISPR-Cas system A is a gene A, and the I-type CRISPR-Cas system A is a modified I-type CRISPR-Cas system; the transformed I type CRISPR-Cas system refers to the I type CRISPR-Cas system which is obtained by transforming gRNA (guide ribonucleic acid) of the I type CRISPR-Cas system in the prior art; the gRNA is transformed into a way that a spacer in the gRNA of an I-type CRISPR-Cas system in the prior art is replaced by a spacer truncation body; and the target gene of the I-type CRISPR-Cas system B is the gene B, and the I-type CRISPR-Cas system is the I-type CRISPR-Cas system in the prior art. According to the invention, gene editing and transcription inhibition of different degrees can be realized at the same time.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for simultaneously realizing gene editing and transcription regulation by utilizing a type I CRISPR-Cas system. Background technique [0002] The CRISPR-Cas system has gained wide application as a gene editing tool and has also been developed for transcriptional regulation. Type I CRISPR-Cas systems are the most widely distributed. The principle of type I CRISPR-Cas system for gene editing is that the gRNA binds to the Cascade effector, and after recognizing the target sequence, Cas3 endonuclease is recruited to cut the target, resulting in double strand break (DSB), activating non- Homologous end joining or homologous recombination repair mechanisms to introduce mutations in target genes or perform precise editing of target genes. To regulate the transcription of target genes, it is usually necessary to knock out the Cas3 protein or mutate its active site to p...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N9/22C12N15/90C12N15/82
CPCC12N15/113C12N9/22C12N15/902C12N15/8261C12N2310/20
Inventor 向华杜开心龚路遥李明
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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