Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen

Abstract

Genome-wide screens that have viability as a readout have been instrumental to identify essential genes. The development of gene knockout screens with the use of CRISPR-Cas has provided a more sensitive method to identify these genes. Here, we performed an exhaustive genome-wide CRISPR/Cas9 phenotypic rescue screen to identify modulators of cytotoxicity induced by the pioneer transcription factor, DUX4. Misexpression of DUX4 due to a failure in epigenetic repressive mechanisms underlies facioscapulohumeral muscular dystrophy (FHSD), a complex muscle disorder that thus far remains untreatable. As the name implies, FSHD generally starts in the muscles of the face and shoulder girdle. Our CRISPR/Cas9 screen revealed no key effectors other than DUX4 itself that could modulate DUX4 cytotoxicity, suggesting that treatment efforts in FSHD should be directed towards direct modulation of DUX4 itself. Our screen did however reveal some rare and unexpected genomic events, that had an important impact on the interpretation of our data. Our findings may provide important considerations for planning future CRISPR/Cas9 phenotypic survival screens.

Functional genetics reveals modulators of anti-microtubule drug sensitivity

“…Although such events likely occur at a low frequency, nocodazole treatment may select for cells with heterozygous loss of the p-arm of chromosome 1 due to the potent suppressive effect of Kif2C-depletion on nocodazole-mediated toxicity. This chromosome arm-loss and suppression behavior has also been observed by others (Ashoti et al, 2022; Cullot et al, 2019) and underscores its relevance for CRISPR screens with potent dose-sensitive suppressors, in which Cas9-mediated chromosome arm cleavage may contribute to false positives. Together, these results demonstrate that the loss of KIF2C acts as a robust suppressor of the microtubule-destabilizing drug nocodazole.…”

Efficient generation of lower induced motor neurons by coupling Ngn2 expression with developmental cues

“…These methods use the intrinsic variability of single nucleotide polymorphisms within a population as a barcode to assign identities in a mixed culture—a “village”—of multiple donors, similarly to pooled CRISPR-Cas9 barcoded screens. 42 – 44 More precisely, Census-seq allows population-scale, quantitative identity assignment from a mixed group of donors, 1 Dropulation can assign identities at a single cell level in a village for scRNA-seq studies. 2 With this aim in mind, we produced liMNs villages: 50 embryonic stem cell lines, previously subjected to whole-genome sequencing, were separately differentiated into liMNs.…”

Proteome-wide systems genetics identifies UFMylation as a regulator of skeletal muscle function

“…Functional screening of genetic perturbations with high-throughput phenotypic measurements have identified novel regulators of muscle biology and disease. These include forward genetic mutagenesis screens to identify regulators of skeletal muscle development and locomotion in zebrafish ( Birely et al, 2005 ; Horstick et al, 2013 ; Johnson et al, 2013 ; Bennett et al, 2018 ) and worms ( Beron et al, 2015 ), RNAi screening of muscle size and function in fruit fly ( Kao et al, 2021 ; Graca et al, 2021 ), and CRISPR/Cas9 screening of muscle cells to identify regulators of myogenesis and cell survival in dystrophy models ( Bi et al, 2017 ; Lek et al, 2020 ; Ashoti et al, 2022 ). Here, we combined forward genetics via proteomic analysis of a diverse mouse panel with a targeted reverse genetics screen via AAV6 vector-mediated expression of shRNAs to knock down specific genes in bioengineered skeletal muscle to identify candidate regulators of skeletal muscle function.…”