A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes

Abstract

Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts.We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

Highly efficient synthetic CRISPR RNA/Cas9-based mutagenesis for rapid cardiovascular phenotypic screening in F0 zebrafish

“…This finding is consistent with a recent study noting variable mutagenic efficiency of crRNAs [37]. We also compared the site of injections (cytoplasm vs yolk) since a recently published work using a similar approach performed injections exclusively into the yolk to examine behavioral phenotypes [32]. Our results showed that although three dgRNP injections into either site were both highly efficient, their cytoplasmic injections were slightly more consistent in phenocopying homozygous stable mutant phenotypes.…”

“…While dgRNP-based mutagenesis utilizing chemically synthesized crRNAs have been shown to efficiently disrupt gene function in F0 zebrafish even as single dgRNP injections [31][32][33][34][35][36][37], it is becoming clear that the efficacy of target gene disruption depends on the mutagenic ability of each designed crRNA, as also suggested by the recent report showing variable efficiency of synthetic crRNAs in gene editing [37]. As such, injection of single dgRNPs per gene can lead to varied mutagenesis efficiency, resulting in variable and incosistent phenotypic observations across crRNAs targeted to the same gene in injected F0 animals.…”

Global analysis of cell behavior and protein localization dynamics reveals region-specific functions for Shroom3 and N-cadherin during neural tube closure

“…F0 mutagenesis using CRISPR has recently emerged as a powerful tool in Xenopus and zebrafish, and mosaic crispants generated by targeted injections allow simultaneous observation of wild-type and crispant cellular phenotypes so that observations are automatically staged and synchronized (Aslan et al, 2017; Kakebeen et al, 2020; Kroll et al, 2021; Szenker-Ravi et al, 2018; Willsey et al, 2020). We therefore labeled the neural plate by injection of fluorescent reporters into both dorsal blastomeres at the 4-cell stage, and then injected a mixture of shroom3 -targeted sgRNA, Cas9 protein, and membrane-BFP mRNA into one dorsal blastomere of 8-cell stage embryos to generate mosaic crispants ( Fig.…”

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

“…We reasoned that directly injecting PCR amplified linear DNA with short homology arms (~35bp) could be highly effective since these donors are relatively small (~780 bp) compared to plasmids (several kbs) , and therefore a small quantity of donors (~10 ng/ul) will provide a large number of molecules (~20 nM) available to engage the HDR machinery following the Cas9 induced DSB. Building on recent improvements in CRISPR/Cas9 knock-in strategies, we used 5' biotinylated primers in order to limit in vivo concatemerization of the donor construct (Gutierrez-Triana et al, 2018), and synthetic sgRNAs were used to increase the efficiency of DSBs by Cas9 (Paix et al, 2015, Kroll et al, 2021, Hoshijima et al, 2019, Li et al, 2019. In addition, we utilized a monomeric streptavidin tagged Cas9 that has a high affinity to the biotinylated donor fragments to increase targeting efficiency (Gu et al, 2018).…”