# US 2019/0376089 A1 · Type i-b crispr-cas system gene cas3-based gene editing method

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2019/0376089 A1
- Document ID: US20190376089A1
- Status: Published application
- Kind: A1
- Application number: 16/487,443
- Filed: 2018-07-24
- Published: 2019-12-12
- Earliest claimed: 2017-09-19
- Assignees: Anhui University
- Applicants: Anhui University
- Inventors: Wangyu TONG; Yanyan TANG; Tingting XIA
- Web page: https://patentgrep.com/patent/US20190376089A1

## CPC

- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/907: {in mammalian cells}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/224: {Class 1 CAS enzyme complex, e.g. multi-enzyme}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A novel gene editing system developed based on the gene cas3 in the type I-B CRISPR-Cas system in the chromosome of Streptomyces virginiae IBL14 enables the gene editing of a type I CRISPR-Cas system on biological cell genomes for the first time. In the system, Cas3 can specifically cleave a DNA fragment of interest guided by crRNA or t-DNA. The new approach can be applied to rapid, simple and correct genome editing of prokaryotic and eukaryotic cells, which provides novel supplements and selections for the commercialized gene editing system developed based on Cas9 in type H.

## Claims

### Claim 1

A type I-B CRISPR-Cas system gene cas3-based gene editing method comprising the following step:
performing a gene editing on genetic materials of all organisms by using a gene editing tool consisting of a protein Cas3 expression vector constructed by the gene cas3 in Streptomyces virginiae IBL14 and a gene editing vector comprising a t-DNA and/or a g-DNA.

### Claim 2 (depends on 1)

The type I-B CRISPR-Cas system gene cas3-based gene editing method according to claim 1, further comprising the following steps:
(1) construction of Cas3 expression vector
designing primers based on a sequence information of the gene cas3 in Streptomyces virginiae IBL14, using a Streptomyces virginiae IBL14 genome as a template, amplifying to obtain the gene cas3 by a PCR reaction, and ligating the gene cas3 to a vector to obtain the Cas3 expression vector;
(2) construction of gene editing vector
(A) designing primers according to a sequence of a gene of interest, using a biological genome of the interest as a template, designing and synthesizing a t-DNA fragment with a desired genetic function by PCR, wherein the t-DNA fragment has upstream and downstream homologous arms of the gene of interest;
(B) designing and chemically synthesizing a g-DNA fragment sequentially consisting of a restriction site, a transcription promoter, a crDNA for transcribing a crRNA, a transcription terminator, and a restriction site according to a sequence information of a biological target gene; and
(C) respectively ligating the t-DNA fragment prepared and the g-DNA fragment to a vector to obtain the gene editing vector;
(3) construction and validation of gene-edited recombinant
preparing competent cells or protoplasts, and introducing the Cas3 expression vector obtained in the step (1) and the gene editing vector obtained in the step (2) into the competent cells or the protoplasts to obtain the gene-edited recombinant; performing PCR and DNA sequencing and/or functional analysis on a recombinant chromosomal gene to confirm the gene-edited recombinant.

### Claim 3 (depends on 1)

The type I-B CRISPR-Cas system gene cas3-based gene editing method according to claim 1, wherein the t-DNA is a chemically and biologically synthesized t-DNA fragment with a desired genetic function, and the chemically and biologically synthesized t-DNA fragment is combined with the Cas3 expression vector or the gene editing vector, or exists independently.

### Claim 4 (depends on 1)

The type I-B CRISPR-Cas system gene cas3-based gene editing method according to claim 1, wherein the g-DNA is a chemically or biologically synthesized g-DNA fragment configured to transcribe the g-DNA into a crRNA, and the chemically or biologically synthesized g-DNA fragment binds into the Cas3 expression vector or the gene editing vector.

### Claim 5 (depends on 1)

The type I-B CRISPR-Cas system gene cas3-based gene editing method according to claim 1, wherein the genetic materials of all organisms are genetic materials originated from prokaryotic cells, or eukaryotic cells, or/and viruses without cellular structures.

### Claim 6 (depends on 1)

The type I-B CRISPR-Cas system gene cas3-based gene editing method according to claim 1, wherein the gene editing is a genome editing mediated through specific cleavage of Cas3 on a target DNA guided by a crRNA or the t-DNA.

### Claim 7 (depends on 1)

The type I-B CRISPR-Cas system gene cas3-based gene editing method according to claim 1, wherein the gene editing is one selected from the group consisting of deletion, insertion, scarless point mutation, and a combination of the deletion, insertion, scarless point mutation performed on biological genetic materials.

### Claim 8

The type I-B CRISPR-Cas system gene cas3-based gene editing method according to 2, wherein the t-DNA is a chemically and biologically synthesized t-DNA fragment with the desired genetic function, and the chemically and biologically synthesized t-DNA fragment is combined with the Cas3 expression vector or the gene editing vector, or exists independently.

### Claim 9 (depends on 2)

The type 1-B CRISPR-Cas system gene cas3-based gene editing method according to claim 2, wherein the g-DNA is a chemically or biologically synthesized g-DNA fragment configured to transcribe to obtain the crRNA, and the chemically or biologically synthesized g-DNA fragment binds into the Cas3 expression vector or the gene editing vector.