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1. WO2021109302 - METHOD FOR PREPARING NEAR-INFRARED LIGHT CONTROL-BASED CRISPR-CAS13A SYSTEM AND APPLICATION THEREOF

Publication Number WO/2021/109302
Publication Date 10.06.2021
International Application No. PCT/CN2019/130227
International Filing Date 31.12.2019
IPC
C12N 15/90 2006.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
90Stable introduction of foreign DNA into chromosome
C12N 9/22 2006.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
9Enzymes, e.g. ligases (6.); Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating, or purifying enzymes
14Hydrolases (3.)
16acting on ester bonds (3.1)
22Ribonucleases
CPC
C12N 15/902
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
90Stable introduction of foreign DNA into chromosome
902using homologous recombination
C12N 2310/20
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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2310Structure or type of the nucleic acid
10Type of nucleic acid
20involving clustered regularly interspaced short palindromic repeats [CRISPRs]
C12N 9/22
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
9Enzymes; Proenzymes; Compositions thereof
14Hydrolases (3)
16acting on ester bonds (3.1)
22Ribonucleases ; RNAses, DNAses
Applicants
  • 江苏大学 JIANGSU UNIVERSITY [CN]/[CN]
Inventors
  • 陈全胜 CHEN, Quansheng
  • 刘蕊 LIU, Rui
  • 李欢欢 LI, Huanhuan
  • 吕鹏 LV, Peng
  • 黄栋 HUANG, Dong
  • 欧阳琴 OUYANG, Qin
Agents
  • 北京德崇智捷知识产权代理有限公司 JW IP LAW FIRM
Priority Data
201911227681.404.12.2019CN
201911343995.024.12.2019CN
Publication Language Chinese (ZH)
Filing Language Chinese (ZH)
Designated States
Title
(EN) METHOD FOR PREPARING NEAR-INFRARED LIGHT CONTROL-BASED CRISPR-CAS13A SYSTEM AND APPLICATION THEREOF
(FR) PROCÉDÉ DE PRÉPARATION D'UN SYSTÈME CRISPR-CAS13A À COMMANDE DE LUMIÈRE PROCHE INFRAROUGE ET APPLICATION ASSOCIÉE
(ZH) 一种基于近红外光控的CRISPR-Cas13a系统制备方法及其应用
Abstract
(EN)
A method for preparing a near-infrared light control-based CRISPR-Cas13a system and an application thereof, the method comprising: CrRNA is designed and synthesized according to the sequence of a target gene mRNA; the crRNA and a purified Cas13a protein form a Cas13a-crRNA compound; the Cas13a-crRNA compound is connected to an up-conversion nanomaterial by means of a photosensitive molecule so as to obtain UCNPs-Cas13a; then the UCNPs-Cas13a is coated using PEI to obtain UCNPs-Cas13a@PEI; the prepared UCNPs-Cas13a@PEI is attached to a surface of a eukaryotic cell and enters the cell, and the PEI of the UCNPs-Cas13a@PEI is used to promote the escape of an endosome; the Cas13a-crRNA is released under the irradiation of an NIR laser; a complementary sequence corresponding to a crRNA spacer sequence is searched for on the target gene mRNA; and crRNA is matched with the target gene mRNA so as to activate a HEPN catalytic site of Cas13a to shear mRNA, and then the expression of a eukaryote target gene is inhibited.
(FR)
L'invention concerne un procédé de préparation d'un système CRISPR-Cas13a à commande de lumière proche infrarouge et une application associée, le procédé comprenant ce qui suit : le crARN est conçu et synthétisé en fonction de la séquence d'un ARNm de gène cible; le crARN et une protéine Cas13a purifiée forment un composé Cas13a-crARN; le composé Cas13a-crARN est relié à un nanomatériau de conversion ascendante au moyen d'une molécule photosensible de façon à obtenir des UCNP-Cas13a; puis les UCNP-Cas13a sont revêtus à l'aide de poly(éthylèneimine) (PEI) pour obtenir des UCNP-Cas13a@PEI; les UCNP-Cas13a@PEI préparés sont fixés à une surface d'une cellule eucaryote et entrent dans la cellule et le PEI des UCNP-Cas13a@PEI est utilisé pour favoriser l'échappement d'un endosome; le Cas13a-crARN est libéré sous l'effet de l'irradiation d'un laser NIR; une séquence complémentaire correspondant à une séquence d'espaceur de crARN est recherchée sur l'ARNm du gène cible; et un crARN est mis en correspondance avec l'ARNm du gène cible de façon à activer un site catalytique HEPN de Cas13a pour cisailler l'ARNm, puis l'expression d'un gène cible d'eucaryote est inhibée.
(ZH)
一种基于近红外光控的CRISPR-Cas13a系统制备方法及其应用,根据目的基因mRNA的序列设计合成crRNA,将其和纯化的Cas13a蛋白形成Cas13a-crRNA复合物,通过光敏分子将Cas13a-crRNA复合物与上转换纳米材料连接在一起获得UCNPs-Cas13a;再用PEI涂覆UCNPs-Cas13a得到UCNPs-Cas13a@PEI;所制备的UCNPs-Cas13a@PEI附着在真核生物细胞表面并进入细胞,利用UCNPs-Cas13a@PEI的PEI促进内体逃逸;在NIR激光照射下Cas13a-crRNA被释放,在目的基因mRNA上寻找与crRNA间隔序列对应的互补序列,crRNA与目的基因mRNA匹配以激活Cas13a的HEPN催化位点剪切mRNA,进而抑制真核生物目的基因表达。
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