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Li, Pan; Zhang, Lijun; Li, Zhifang; Xu, Chunlong; Du, Xuguang; Wu, Sen
Cellular and molecular life sciences : CMLS, 10/2020, Letnik: 77, Številka: 19Journal Article
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.
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JCR | SNIP | JCR | SNIP | JCR | SNIP | JCR | SNIP |
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