Shota Nakade

TL;DR: It is demonstrated that CRISPR/Cas9-mediated PITCh, termed CRIS-PITCh, can be applied in human cells without carrying the plasmid backbone sequence and will be useful for a variety of applications, not only in cultured cells, but also in various organisms, including invertebrates and vertebrates.

TL;DR: A streamlined protocol for PITCh knock-in is described, 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.

TL;DR: Efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences flanking the genomic target locus enables precise targeted gene knock-in in zebrafish.

TL;DR: Various improvements and alternatives of CRISPR-Cas systems are described, including engineered Cas9 variants, Cas9 homologs, and novel Cas proteins other than Cas9 that enable flexible genome engineering with high efficiency and specificity, orthogonal genetic control at multiple gene loci, gene knockdown, or fluorescence imaging of transcripts mediated by RNA targeting, and beyond.

TL;DR: This work identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells and mouse zygotes by combining the Exo1 and PITCh-directed donor vectors, which provide a technical platform for high-throughput knock-in.