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  • Highly efficient and safe genome editing by CRISPR-Cas12a using CRISPR RNA with a ribosyl-2'-O-methylated uridinylate-rich 3'-overhang in mouse zygotes.

Highly efficient and safe genome editing by CRISPR-Cas12a using CRISPR RNA with a ribosyl-2'-O-methylated uridinylate-rich 3'-overhang in mouse zygotes.

Experimental & molecular medicine (2020-11-10)
Dae-In Ha, Jeong Mi Lee, Nan-Ee Lee, Daesik Kim, Jeong-Heon Ko, Yong-Sam Kim
ABSTRACT

The CRISPR-Cas12a system has been developed to harness highly specific genome editing in eukaryotic cells. Given the relatively small sizes of Cas12a genes, the system has been suggested to be most applicable to gene therapy using AAV vector delivery. Previously, we reported that a U-rich crRNA enabled highly efficient genome editing by the CRISPR-Cas12a system in eukaryotic cells. In this study, we introduced methoxyl modifications at C2 in riboses in the U-rich 3'-overhang of crRNA. When mixed with Cas12a effector proteins, the ribosyl-2'-O-methylated (2-OM) U-rich crRNA enabled improvement of dsDNA digestibility. Moreover, the chemically modified U-rich crRNA achieved very safe and highly specific genome editing in murine zygotes. The engineered CRISPR-Cas12a system is expected to facilitate the generation of various animal models. Moreover, the engineered crRNA was evaluated to further improve a CRISPR genome editing toolset.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Hyaluronidase from bovine testes, Type I-S, lyophilized powder, 400-1000 units/mg solid
Sigma-Aldrich
M2 medium, With HEPES, without penicillin and streptomycin, liquid, sterile-filtered, suitable for mouse embryo cell culture
Sigma-Aldrich
EmbryoMax® KSOM Mouse Embryo Media, (1X), Liquid, with 1/2 Amino Acids & Phenol Red