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MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems

Abstract

Programmable nucleases enable engineering of the genome by utilizing endogenous DNA double-strand break (DSB) repair pathways. Although homologous recombination (HR)-mediated gene knock-in is well established, it cannot necessarily be applied in every cell type and organism because of variable HR frequencies. We recently reported an alternative method of gene knock-in, named the PITCh (Precise Integration into Target Chromosome) system, assisted by microhomology-mediated end-joining (MMEJ). MMEJ harnesses independent machinery from HR, and it requires an extremely short homologous sequence (5–25 bp) for DSB repair, resulting in precise gene knock-in with a more easily constructed donor vector. Here we describe a streamlined protocol for PITCh knock-in, including the design and construction of the PITCh vectors, and their delivery to either human cell lines by transfection or to frog embryos by microinjection. The construction of the PITCh vectors requires only a few days, and the entire process takes 1.5 months to establish knocked-in cells or 1 week from injection to early genotyping in frog embryos.

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Figure 1: A simplified schematic of DSB repair mechanisms induced by TALENs and CRISPR-Cas9.
Figure 2: General outlines of HR-, NHEJ- and MMEJ-mediated gene knock-in.
Figure 3: A schematic of TAL-PITCh-mediated whole plasmid integration.
Figure 4: The original and modified CRIS-PITCh systems (v1 and v2) for cassette knock-in.
Figure 5: General workflow of CRIS-PITCh (v2)-mediated gene knock-in in cultured cells.
Figure 6: In vivo visualization of endogenous keratin protein fused to EGFP in Xenopus laevis16.
Figure 7: A schematic illustration of vector construction for CRIS-PITCh (v2)–mediated gene knock-in in cultured cells.
Figure 8: Options for constructing the TAL-PITCh donor vector.
Figure 9: CRIS-PITCh (v2)–mediated cassette knock-in in HEK293T cells.

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Acknowledgements

The authors express their appreciation to A. Kawahara and Y. Hisano (University of Yamanashi, Yamanashi, Japan) for co-developing the modified PITCh system. We also thank H. Ochiai (Hiroshima University, Hiroshima, Japan) for sharing the synthesized mNeonGreen cDNA under the license agreement with Allele Biotechnology and Pharmaceuticals, Inc. This work was supported by the Japan Society for the Promotion of Science (25890014 to T.S., 25124708 to K.-I.T.S. and 26290070 to T.Y.), the Sasakawa Foundation (to S.N.), the Uehara Memorial Foundation (to T.S.) and the Ministry of Health, Labor, and Welfare of Japan (to T.Y.).

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T.S. organized and wrote the manuscript. S.N. performed the human cell experiments and wrote the manuscript concerning human cell procedures. Y.S. performed the frog experiments. K.-I.T.S. wrote the manuscript concerning frog procedures. T.Y. supervised the work.

Corresponding author

Correspondence to Tetsushi Sakuma.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Design and validation of the PITCh-gRNAs.

Three gRNAs were initially designed, and safety and efficacy validated by online off-target searches and single-strand annealing (SSA) assays1, respectively. (a) Target sequence of each artificially-designed PITCh-gRNA. PAM sequence is underlined. (b) In silico validation of each PITCh-gRNA using the CRISPR design tool (http://crispr.mit.edu/). Higher scores indicate lower off-target risks in corresponding organisms. (c) Experimental validation of DSB-inducing activities by human cell-based SSA assay. pSTL-ZFA36 vector1 was used for the positive control zinc-finger nuclease (ZFN). Blue bars indicate negative control samples that reporter vectors harboring unrelated sequence were introduced. Red bars indicate test samples that reporter vectors harboring corresponding target sequence were introduced.

1. Ochiai, H. et al. Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases. Genes Cells 15, 875–885 (2010).

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Sakuma, T., Nakade, S., Sakane, Y. et al. MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nat Protoc 11, 118–133 (2016). https://doi.org/10.1038/nprot.2015.140

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