Establishment of expanded and streamlined pipeline of PITCh knock-in – a web-based design tool for MMEJ-mediated gene knock-in, PITCh designer, and the variations of PITCh, PITCh-TG and PITCh-KIKO

Abstract

The emerging genome editing technology has enabled the creation of gene knock-in cells easily, efficiently, and rapidly, which has dramatically accelerated research in the field of mammalian functional genomics, including in humans. We recently developed a microhomology-mediated end-joining-based gene knock-in method, termed the PITCh system, and presented various examples of its application. Since the PITCh system only requires very short microhomologies (up to 40 bp) and single-guide RNA target sites on the donor vector, the targeting construct can be rapidly prepared compared with the conventional targeting vector for homologous recombination-based knock-in. Here, we established a streamlined pipeline to design and perform PITCh knock-in to further expand the availability of this method by creating web-based design software, PITCh designer (http://www.mls.sci.hiroshima-u.ac.jp/smg/PITChdesigner/index.html), as well as presenting an experimental example of versatile gene cassette knock-in. PITCh designer can automatically design not only the appropriate microhomologies but also the primers to construct locus-specific donor vectors for PITCh knock-in. By using our newly established pipeline, a reporter cell line for monitoring endogenous gene expression, and transgenesis (TG) or knock-in/knockout (KIKO) cell line can be produced systematically. Using these new variations of PITCh, an exogenous promoter-driven gene cassette expressing fluorescent protein gene and drug resistance gene can be integrated into a safe harbor or a specific gene locus to create transgenic reporter cells (PITCh-TG) or knockout cells with reporter knock-in (PITCh-KIKO), respectively.

Bioengineering tools for the production of pharmaceuticals: current perspective and future outlook

“…Nakamae et al [19] have been described as a newly advanced microhomology intermediated end assembly dependent gene knocks tool, named the PITCh tool, and offered numerous applications. Ventura et al [20] have described the probiogenomics as a new tool to get genetic perceptions into a variation of probiotic prokaryotes into the human guts.…”

“…Available bioengineered tools and techniques for the production of pharmaceuticals. [19] not only the appropriate micro homologies but also the primers to construct locus-specific donor vectors for PITCh knock-in. By using the newly established pipelines, a reporter cell line for monitoring endogenous gene expression, and transgenesis (TG) or knock-in/knockout (KIKO) cell line can be produced systematically [19].…”

CRISPR/Cas9-mediated integration of large transgene into pig potential safe harbor

“…2015; Sakuma et al . 2016; Nakamae et al . 2017), but there is still no success for targeted integration of large fragments over 20 kbp.…”

Magic wands of CRISPR—lots of choices for gene knock-in

“…In addition, the detailed protocol, freely available materials, and online design tool for the CRISPR-mediated PITCh knock-in were provided by the authors Nakamae et al 2017). Along with these user-friendly resources, the application examples of CRISPR-mediated PITCh have become more diversified, e.g., high-throughput epitope tagging in cultured cells for chromatin immunoprecipitation (ChIP)-seq (Xiong et al 2017), the application in mammalian zygotes (Aida et al 2016;Nakagawa et al 2017), and in vivo somatic knock-in in mice (Yao et al 2017a) were reported one after another.…”