TY - JOUR T1 - Engineering improved Cas13 effectors for targeted post-transcriptional regulation of gene expression JF - bioRxiv DO - 10.1101/2021.05.26.445687 SP - 2021.05.26.445687 AU - Emeric J Charles AU - Shin Eui Kim AU - Gavin J. Knott AU - Dylan Smock AU - Jennifer Doudna AU - David F. Savage Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/05/26/2021.05.26.445687.abstract N2 - Cas13 is a family of unique RNA-targeting CRISPR-Cas effectors, making it an appealing tool for probing and perturbing RNA function. However only a few Cas13 homologs have been shown to mediate robust RNA targeting in human cells, suggesting that unknown elements may be limiting their efficacy. Furthermore, many Cas13 enzymes show high degrees of toxicity upon targeting and have not been shown to mediate specific knockdown in other cell types such as E. coli. Here, we show that catalytically inactive Cas13 enzymes can be repurposed for efficient translational repression in bacteria with no associated growth defects. To achieve this advance, we carried out a directed evolution screen to engineer functional Cas13a variants, and identified a number of stabilizing mutations, which enabled efficient post transcriptional knockdown of gene expression. In vitro characterization of the resulting engineered Lbu Cas13a mutant, termed eLbu, revealed both stabilization and altered cleavage kinetics. Finally, we show that eLbu can be used for efficient exon skipping in human cells. This work represents the first demonstration of targeted translational repression in E. coli using a CRISPR enzyme, as well as the first directed evolution of a Cas13 enzyme. Such a platform could allow for engineering other aspects of this protein family to obtain more robust RNA targeting tools.Competing Interest StatementJ.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Scribe Therapeutics, and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Caribous Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Synthego, Metagenomi, Mammoth Biosciences, and Inari. J.A.D is a member of the board of directors at Driver and Johnson & Johnson. D.F.S. is a co-founder of Scribe Therapeutics and a scientific advisory board member of Scribe Therapeutics and Mammoth Biosciences. All other authors declare no competing interests. ER -