Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish

Abstract

Many eukaryotic genes play essential roles in multiple biological processes in several different tissues. Conditional mutants are needed to analyze genes with such pleiotropic functions. In vertebrates, conditional gene inactivation has only been feasible in the mouse, leaving other model systems to rely on surrogate experimental approaches such as overexpression of dominant negative proteins and antisense-based tools. Here, we have developed a simple and straightforward method to integrate loxP sequences at specific sites in the zebrafish genome using the CRISPR/Cas9 technology and oligonucleotide templates for homology directed repair. We engineered conditional (floxed) mutants of tbx20 and fleer, and demonstrate excision of exons flanked by loxP sites using tamoxifen-inducible CreERT2 recombinase. To demonstrate broad applicability of our method, we also integrated loxP sites into two additional genes, aldh1a2 and tcf21. The ease of this approach will further expand the use of zebrafish to study various aspects of vertebrate biology, especially post-embryonic processes such as regeneration.

Model systems for regeneration: zebrafish

“…These have proven useful for generating null mutants in zebrafish (Hwang et al, 2013;Jao et al, 2013) and for assessing gene function during regeneration (Dupret et al, 2017;Dong et al, 2019;Nauroy et al, 2019;Unal Eroglu et al, 2018). The generation of conditional knockouts has also recently been achieved by the directed insertion of loxP sites using CRISPR/Cas9 (Burg et al, 2018). This new approach will be extremely valuable to address gene function during regeneration.…”

Deletion of MOrpholino Binding Sites (DeMOBS) to Assess Specificity of Morphant Phenotypes

“…Two S. pyogenes PAM sites are present within the 5'UTR sequence targeted by tbx5a-MO4 (Lu et al, 2008) (Figure 1B). We synthesized 19-base guide RNAs with a G nucleotide (lower case in Figure 1B) required for transcription initiation by the T7 RNA polymerase added, and injected them along with nCas9n mRNA as previously described (Burg et al, 2018;Burg et al, 2016). PCR fragments amplified on lysates from 20 pooled 3 day post fertilization (dpf) injected embryos were analyzed for sgRNA efficiency by TIDE (Brinkman et al, 2014) and Synthego ICE (Hsiau et al, 2019).…”

“…While the mutagenesis method employed by us is likely feasible for the majority of MOs targeting 5' UTRs, there will inevitably be a subset where PAM sites will be absent or located closer to the middle or the 3' of the target site. In such cases, oligonucleotide-mediated repair of double strand breaks can be used to engineer desired mutations (Bedell et al, 2012;Burg et al, 2018;Burg et al, 2016;Dong et al, 2014;Gagnon et al, 2014;Gibb et al, 2018;Hruscha et al, 2013;Prykhozhij et al, 2018).…”

“…Guide RNAs were produced as previously described (Burg et al, 2018;Burg et al, 2016) using DR274 (Hwang et al, 2013) as the template and diluted to ~60 ng/ μL. Immediately prior to injection, 8 uL of diluted sgRNA was mixed with 2 μL aliquot of 150 ng/ μL nCas9n mRNA (Jao et al, 2013) to the final volume of 10 μL.…”

“…With the notable exception of short upstream reading frames (Johnstone et al, 2016), 5' UTRs appear to be sparse in significant regulatory features. We speculated that indel mutations within 5' UTRs are unlikely to significantly impair the expression of the downstream gene (Burg et al, 2018). Introduced indels within a morpholino target site should reduce, if not entirely abolish, MO binding making the "mutant mRNA" partly or completely refractive to morpholino binding.…”