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Cas12a mediates efficient and precise endogenous gene tagging via MITI: microhomology-dependent targeted integrations.

ABSTRACT: Efficient exogenous DNA integration can be mediated by Cas9 through the non-homology end-joining pathway. However, such integrations are often imprecise and contain a variety of mutations at the junctions between the external DNA and the genomic loci. Here we describe a microhomology-dependent targeted integration method, designated MITI, for precise site-specific gene insertions. We found that the MITI strategy yielded higher knock-in accuracy than Cas9 HITI for the insertion of external DNA and tagging endogenous genes. Furthermore, in combination with negative selection and four different CrRNAs targeting donor vectors and genome-targeted sites with a CrRNA array, MITI facilitated precise ligation at all junctions. Therefore, our Cas12a-based MITI method increases the repertoire of precision genome engineering approaches and provides a useful tool for various gene editing applications.

SUBMITTER: Li P

PROVIDER: S-EPMC7508734 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Cas12a mediates efficient and precise endogenous gene tagging via MITI: microhomology-dependent targeted integrations.

Li Pan P Zhang Lijun L Li Zhifang Z Xu Chunlong C Du Xuguang X Wu Sen S

Cellular and molecular life sciences : CMLS 20191217 19

Efficient exogenous DNA integration can be mediated by Cas9 through the non-homology end-joining pathway. However, such integrations are often imprecise and contain a variety of mutations at the junctions between the external DNA and the genomic loci. Here we describe a microhomology-dependent targeted integration method, designated MITI, for precise site-specific gene insertions. We found that the MITI strategy yielded higher knock-in accuracy than Cas9 HITI for the insertion of external DNA an ...[more]

PMID: 31848638

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