Our paper "Optimizing gRNA selection for high-penetrance F0 CRISPR screening for interrogating disease gene function" is now published in @narjournal.bsky.social doi.org/10.1093/nar/.... This work was led by our super postdoc @sheng-jia.bsky.social

Several papers on F0 mutagenesis have been published, including those by Jao et al., Burger et al., Wu et al., Kroll et al., and others—all methods work well. We set out to test and compare strategies to achieve the highest phenotype penetrance using minimal resources.

We utilized indel prediction models such as Lindel (or inDelphi/ForeCast and others) to design gRNAs with high predicted success. Targeting the N-terminal half of functional domains worked best. We tested 324 gRNAs across 125 genes, targeting key protein domains with just 1-2 gRNAs.

Our data shows that targeting the N-terminal half of a functional protein domain significantly increases phenotypic penetrance compared to targeting other regions of the gene. Selecting gRNAs with high Lindel scores (>75) substantially improved knockout efficiency.

Transcriptomic analysis revealed that gene expression patterns in F0 knockouts closely mimic those in stable knockout lines.