Genome-scale CRISPR screening in a single mouse liver

Abstract

Our ability to understand and modulate mammalian physiology and disease requires knowing how all genes contribute to any given phenotype in the organism. Genome-wide screening using CRISPR-Cas9 has emerged as a powerful method for the genetic dissection of cellular processes1,2, but the need to stably deliver single guide RNAs to millions of cells has restricted its implementation to ex vivo systems. These ex vivo systems cannot reproduce all of the cellular phenotypes observed in vivo nor can they recapitulate all of the factors that influence these phenotypes. There thus remains a pressing need for high-throughput functional genomics in a living organism. Here, we establish accessible genome-wide screening in the mouse liver and use this approach to uncover the complete regulation of cellular fitness in a living organism. We discover novel sex-specific and cell non-autonomous regulation of cell growth and viability. In particular, we find that the class I major histocompatibility complex is essential for preventing immune-mediated clearance of hepatocytes. Our approach provides the first comprehensive picture of cell fitness in a living organism and highlights the importance of investigating cellular phenomena in their native context. Our screening method is robust, scalable, and easily adapted to examine diverse cellular processes using any CRISPR application. We have hereby established a foundation for high-throughput functional genomics in a living mammal, enabling unprecedented insight into mammalian physiology and disease.

In vivo Pooled Screening: A Scalable Tool to Study the Complexity of Aging and Age-Related Disease

“…Some in vivo screens have achieved efficient delivery using lentivirus administered during development ( Shah et al, 2015 ; Jin et al, 2020 ), but using this approach to study aging would be very slow. Similarly, some screens have combined mice engineered to express Cas9 with guide delivery via AAV ( Chow et al, 2017 ) or lentivirus ( Wertz et al, 2020 ; Keys and Knouse 2021 ). Cas9 effector-expressing mice are much more versatile than genetically engineered models, because a single mouse line can be combined with different gRNA libraries to perform a variety of screens ( Lima and Maddalo 2021 ).…”

Brap regulates liver morphology and hepatocyte turnover via modulation of the Hippo pathway

“…These findings raise the exciting possibility that glycogen could play a role in mediating the BRAP-MST2 interaction. CRISPR screening has identified Brap as one of a select few sex-specific essential genes in developing mouse livers; our own observations indicate that male Brap LKO mice display more dramatic changes in liver structure than do female mice ( 21 ). Thus, the sex-specific roles of Brap also invite future inquiry.…”