Protocol for in vivo CRISPR screening targeting murine testicular cells
Year: 2024
Type: article
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 • Protocol for in vivo genome-wide screening targeting murine testicular cells • Method for introducing lentivirus-based sgRNA library into male germ cells • Instructions for applying the revival screening method to testis • Pipeline for analyzing sgRNA library results Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.
Source: STAR Protocols
Authors: Yuki Noguchi, Masahiro Maruoka, Jun Suzuki
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,500
OpenAlex ID: W4402482808