Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

Kim, H.; Lee, W.-j.; Kang, S.-H.; Hur, J. K.; Lee, H.; Song, W.; Lim, K.-S.; Park, Y.-H.; Song, B.-S.; Jin, Y. B.; Jun, B.-H.; Lee, D.-S.; Kim, S.-U.; Lee, S. H.

doi:10.1101/2020.02.04.933614

bioRxiv · 10.1101/2020.02.04.933614

Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

Kim, H.·Lee, W.-j.·Kang, S.-H.·Hur, J. K.·Lee, H.·Song, W.·Lim, K.-S.·Park, Y.-H.·Song, B.-S.·Jin, Y. B.·Jun, B.-H.·Lee, D.-S.·Kim, S.-U.·Lee, S. H.

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Abstract

The CRISPR-Cas9 system is widely used for target-specific genome engineering. Cpf1 is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cpf1 has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cpf1 activity is improved for therapeutic purposes. In our study, we investigated off-target cleavage by Cpf1 and modified the Cpf1 (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cpf1 that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cpf1 and SpCas9 nickase effectively work in the intracellular genome is suggested. In our results, CRISPR-Cpf1 induces less off-target mutations at the cell level, when chimeric DNA-RNA guide was used for genome editing. This study has a potential for therapeutic applications in incurable diseases caused by genetic mutation.

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Kim, H., Lee, W.-j., Kang, S.-H., Hur, J. K., Lee, H., Song, W., Lim, K.-S., Park, Y.-H., Song, B.-S., Jin, Y. B., Jun, B.-H., Lee, D.-S., Kim, S.-U., Lee, S. H.. 2020-02-04. Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide. https://doi.org/10.1101/2020.02.04.933614

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