Abstract
Identifying genes involved in biological processes is critical for understanding the molecular building blocks of life. We used engineered CRISPR (clustered regularly interspaced short palindromic repeats) to efficiently mutate specific loci in zebrafish (Danio rerio) and screen for genes involved in vertebrate biological processes. We found that increasing CRISPR efficiency by injecting optimized amounts of Cas9-encoding mRNA and multiplexing single guide RNAs (sgRNAs) allowed for phenocopy of known mutants across many phenotypes in embryos. We performed a proof-of-concept screen in which we used intersecting, multiplexed pool injections to examine 48 loci and identified two new genes involved in electrical-synapse formation. By deep sequencing target loci, we found that 90% of the genes were effectively screened. We conclude that CRISPR can be used as a powerful reverse genetic screening strategy in vivo in a vertebrate system.
Highlights
While classical forward and reverse genetic approaches have identified key molecular pathways required for life they are generally limiting in the number of targets that can be assessed and are very time intensive
Injection of Cas[9] and a single guide RNA can reproduce known mutant phenotypes[6], suggesting that new genes involved in a process could be identified in injected (F0) embryos or larvae
We show that CRISPR can be used as a reverse genetic screening tool to identify new genes for a wide range of biological processes in zebrafish
Summary
While classical forward and reverse genetic approaches have identified key molecular pathways required for life they are generally limiting in the number of targets that can be assessed and are very time intensive. The DNA break is repaired by one of two cellular mechanisms: homology directed repair that creates a precise copy of a complementary sequence, or non-homologous end joining, which often introduces insertion and deletions (InDels) that can disrupt gene function[3,4]. The latter type of repair has been used in many systems to introduce mutations into genes of interest that, once fixed and carried in the genome, can be assessed for their effect on a process of interest[3,4]. Because of the ease of engineering the targeting specificity of CRISPRs we wanted to examine whether the system could be used as an efficient reverse genetic screening tool to assess many genes for their function in processes of interest in zebrafish
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