Protocol for in vivo CRISPR screening targeting murine testicular cells

Yuki Noguchi; Masahiro Maruoka; Jun Suzuki

doi:10.1016/j.xpro.2024.103306

Protocol for in vivo CRISPR screening targeting murine testicular cells

STAR Protoc. 2024 Sep 11;5(3):103306. doi: 10.1016/j.xpro.2024.103306. Online ahead of print.

Authors

Yuki Noguchi¹, Masahiro Maruoka², Jun Suzuki³

Affiliations

¹ Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyoku, Kyoto 606-8501, Japan; Graduate School of Biostudies, Kyoto University, Konoe-cho, Yoshida, Sakyoku, Kyoto 606-8501, Japan.
² Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyoku, Kyoto 606-8501, Japan.
³ Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyoku, Kyoto 606-8501, Japan; Graduate School of Biostudies, Kyoto University, Konoe-cho, Yoshida, Sakyoku, Kyoto 606-8501, Japan; Center for Integrated Biosystems, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan. Electronic address: jsuzuki@icems.kyoto-u.ac.jp.

PMID: 39269899
DOI: 10.1016/j.xpro.2024.103306

Abstract

In vivo genome-wide screening elucidates tissue-specific molecular events. Here, we present a protocol for an in vivo genome-wide CRISPR-Cas9 single-guide RNA (sgRNA) library screening technique optimized for mouse testicular cells to investigate spermatogenesis. We describe steps for virus injection, sperm sorting, and primase-based whole-genome amplification. We then detail procedures for library reconstruction using a "revival screening" technique. Our approach reveals intricate spermatogenesis processes and is adaptable for diverse tissue-specific studies. For complete details on the use and execution of this protocol, please refer to Noguchi et al.¹.

Keywords: CRISPR; Genomics; Molecular Biology.