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

Pan Li,Xuguang Du,Zhifang Li,Sen Wu,Chunlong Xu,Lijun Zhang

doi:10.1007/s00018-019-03396-8

Pan Li, Xuguang Du + Show 4 more

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https://doi.org/10.1007/s00018-019-03396-8

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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.

Highlights

Site-specific transgene integration is primarily implemented by homology-directed repair (HDR) and non-homology end joining (NHEJ) pathways [1]
Accurate site-specific gene insertions could be mediated via a method called Obligate Ligation-Gated Recombination (ObLiGaRe) through the efficient NHEJ pathway using zinc-finger nucleases (ZFNs) or Tale nucleases (TALENs)
After Sanger sequencing the TA-cloned 5′- and 3′-junction PCR products of the Cas12a homology-independent target integration (HITI), Cas12a microhomology-dependent targeted integration (MITI), and Cas9 HITI HeLa cells, we found that 70% of Cas12a MITI-mediated integrations were accurate at the 5′ junctions, whereas precision of Cas9 HITI integrations was only 16.67%, and Cas12a HITI had no precise integrations (Figs. 2b, S2A-D)

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Introduction

Site-specific transgene integration is primarily implemented by homology-directed repair (HDR) and non-homology end joining (NHEJ) pathways [1]. The precise introduction of foreign DNA to endogenous target sites through the HDR pathway is time-consuming and cumbersome as it requires the cloning of the homology arms for every gene. Since this method can only exert its effect in the S/G2 cell phase, its efficiency is low, especially in non-dividing cells [2]. Accurate site-specific gene insertions could be mediated via a method called Obligate Ligation-Gated Recombination (ObLiGaRe) through the efficient NHEJ pathway using zinc-finger nucleases (ZFNs) or Tale nucleases (TALENs). Several efforts have aimed to optimize this system, such as controlling the expression of Cas protein via fusion of FKBP12-L106P destabilization domain to Cas9 [9] and cleaving both donor and target sites with the same sgRNA via homology-independent target integration (HITI) strategy [10]

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