Comparative landscape of genetic dependencies in human and chimpanzee stem cells

Generation and characterization of inducible KRAB-dCas9 iPSCs from primates for cross-species CRISPRi

“…While the AAVS1 locus has been well established in human cells and has also been used as a target site in some primates, 30 , 39 , 40 in this study, we show the utility of transgene integration at the AAVS1 locus in additional non-human primate iPSCs. More precisely, we present the generation and characterization of inducible KRAB-dCas9 iPSCs of the three primate species human ( Homo sapiens ), gorilla ( Gorilla gorilla ), and cynomolgus ( Macaca fascicularis ).…”

“…Powerful methodologies such as generation of iPSCs in combination with functional genomic readouts have added exciting possibilities to this approach in recent years, 2 , 16 , 19 including the possibility to perform genome-wide gene perturbations by CRISPR screens. 30 Key to such an approach is generating comparable, i.e., homologous cell types across a sufficient number of biological replicates per species. 16 , 18 However, iPSCs are known to differ (epi)genetically among individuals, among iPSC clones of the same individual and among cells within a clone.…”

“…Hence e.g., pro-survival adaptations such as deletions in p53 63 can be easily mistaken as species differences. 30 As genetic modification and clonal selection are necessary to generate stable, nuclease-expressing cells for CRISPR screens, the knock-in process could add additional, unwanted variation that one needs to control for. 64 While isogenic iPSCs that share a single genetic background are a potential solution to control for much of this unwanted variation, this is obviously not possible when working with different species.…”

“… 27 , 28 , 29 Using a CRISPRi screen with gRNA counting as readout, recently revealed unexpected differences in gene dependencies affecting cell cycle control and cell proliferation between human and chimpanzee, exemplifying the relevance of primate comparisons to understand human evolution. 30 CRISPR screens that use gRNA abundance as readout are efficient for studying mechanisms that influence phenotypes like cell survival or proliferation, but high-content readouts such as single-cell RNA-sequencing (scRNA-seq) offer even more complex phenotypic information. 22 While numerous remarkable single-cell CRISPR screens have been performed in individual species, 31 , 32 , 33 , 34 , 35 , 36 cross-species approaches would provide unique additional and functionally relevant information.…”