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

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. Genome editing: chemical change improves CRISPR tool A chemical modification to the CRISPR-Cas12a gene-editing system could allow for safer and more efficient genome editing. Yong-Sam Kim and colleagues from the Korea Research Institute of Bioscience & Biotechnology (KRIBB) in Daejeon, South Korea, chemically altered the RNA molecule that helps guide Cas12a enzymes to their target genome sequences, where the CRISPR machinery initiates DNA cutting. Building on a previous modification, in which Kim’s team at the KRIBB Genome Editing Research Center added poly-uridinylates to the RNA, the researchers further modified the RNA with methyl tags. This increased the DNA-cutting activity of Cas12a, both in test tube experiments and in mouse embryos, resulting in safe and specific genome editing. The modifications could lead to more precise genetic models of human diseases or be incorporated into future gene therapies.