Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virusDo Yon Kim, Jeong Mi Lee, Su Bin Moon et al.|Nature Biotechnology|2021 Gene therapy would benefit from a miniature CRISPR system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in eukaryotic cells. One of the most compact CRISPR-associated nucleases yet discovered is the archaeal Un1Cas12f1. However, Un1Cas12f1 and its variants have very low activity in eukaryotic cells. In the present study, we redesigned the natural guide RNA of Un1Cas12f1 at five sites: the 5' terminus of the trans-activating CRISPR RNA (tracrRNA), the tracrRNA-crRNA complementary region, a penta(uridinylate) sequence, the 3' terminus of the crRNA and a disordered stem 2 region in the tracrRNA. These optimizations synergistically increased the average indel frequency by 867-fold. The optimized Un1Cas12f1 system enabled efficient, specific genome editing in human cells when delivered by plasmid vectors, PCR amplicons and AAV. As Un1Cas12f1 cleaves outside the protospacer, it can be used to create large deletions efficiently. The engineered Un1Cas12f1 system showed efficiency comparable to that of SpCas9 and specificity similar to that of AsCas12a.
Highly efficient genome editing by CRISPR-Cpf1 using CRISPR RNA with a uridinylate-rich 3′-overhangSu Bin Moon, Jeong Mi Lee, Jeong Gu Kang et al.|Nature Communications|2018 Abstract Genome editing has been harnessed through the development of CRISPR system, and the CRISPR from Prevotella and Francisella 1 (Cpf1) system has emerged as a promising alternative to CRISPR-Cas9 for use in various circumstances. Despite the inherent multiple advantages of Cpf1 over Cas9, the adoption of Cpf1 has been unsatisfactory because of target-dependent insufficient indel efficiencies. Here, we report an engineered CRISPR RNA (crRNA) for highly efficient genome editing by Cpf1, which includes a 20-base target-complementary sequence and a uridinylate-rich 3′-overhang. When the crRNA is transcriptionally produced, crRNA with a 20-base target-complementary sequence plus a U 4 AU 4 3′-overhang is the optimal configuration. U-rich crRNA also maximizes the utility of the AsCpf1 mutants and multiplexing genome editing using mRNA as the source of multiple crRNAs. Furthermore, U-rich crRNA enables a highly safe and specific genome editing using Cpf1 in human cells, contributing to the enhancement of a genome-editing toolbox.
Hypercompact adenine base editors based on a Cas12f variant guided by engineered RNADo Yon Kim, Yuhee Chung, Yujin Lee et al.|Nature Chemical Biology|2022 Highly efficient and safe genome editing by CRISPR-Cas12a using CRISPR RNA with a ribosyl-2′-O-methylated uridinylate-rich 3′-overhang in mouse zygotesDae-In Ha, Jeong Mi Lee, Nan‐Ee Lee et al.|Experimental & Molecular Medicine|2020 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.
Author Correction: Hypercompact adenine base editors based on a Cas12f variant guided by engineered RNADo Yon Kim, Yuhee Chung, Yujin Lee et al.|Nature Chemical Biology|2023