Cas12a mediates efficient and precise endogenous gene tagging via MITI: microhomology-dependent targeted integrations
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Cellular and Molecular Life Sciences
ORIGINAL ARTICLE
Cas12a mediates efficient and precise endogenous gene tagging via MITI: microhomology‑dependent targeted integrations Pan Li1,2 · Lijun Zhang1,2 · Zhifang Li1,2 · Chunlong Xu3 · Xuguang Du1,2 · Sen Wu1,2 Received: 22 August 2019 / Revised: 4 November 2019 / Accepted: 26 November 2019 © The Author(s) 2019
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 sitespecific 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. Keywords CRISPR · Genome editing · Homology-independent targeted integration · Negative selection · Sticky ends · Microhomology-dependent targeted integration Abbreviations MITI Microhomology-dependent targeted integration HITI Homology-independent targeted integration CRISPR Clustered regularly interspaced short palindromic repeats Pan Li and Lijun Zhang contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00018-019-03396-8) contains supplementary material, which is available to authorized users. * Xuguang Du [email protected] * Sen Wu [email protected] 1
Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China
2
State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China
3
Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
CrRNAs CRISPR RNAs DSB Double-strand break NHEJ Non-homology end joining HDR Homology-directed repair GOI Gene-of-interest ZFNs Zinc finger nucleases ObLiGaRe Obligate ligation-gated recombination TALENs Tale nucleases PFFs Pig fetal fibroblasts HSV-tk Herpes simplex virus thymidine kinase
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 p
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