CRISPR/Cas9-mediated integration of loxP sequences for conditional mutagenesis in zebrafish has recently made its way into the toolkit of zebrafish researchers1,4. In particular, CRISPR/Cas9 mutagenesis coupled with ssODN (single stranded oligodeoxynucleotide)-mediated repair has simplified experimental design and provided a robust framework for conditional mutagenesis, as readily demonstrated by several groups in the zebrafish community to date2,3. Noted by Burg et al., one challenge in generating these mutant lines lies in the screening process, namely in substantial variability in PCR detection of loxP site integration at target loci.
Indeed, we have noted similar difficulties with detection of loxP insertion events in our own experiments, which have generally required more locus-specific optimizations than other, more standard PCR-based detection assays. A second consideration, emphasized by Burg et al. as well as Boel et al., is in the necessity for sequencing all potential founders to confirm the presence or absence of imprecise ssODN integration events, including complex rearrangements and indel insertions around the loxP sequence itself.
In fact, we have turned to sequential integration of loxP sequences in two stages of injection and screening to circumvent both large deletions across loxP target loci as well as the homology-independent integration of repair templates at opposing target sites, as observed when we attempted to target and edit both loci simultaneously.
Figure 1. The gel image is from a heterozygous incross of germline founders carrying a 5′ loxP insertion
One additional consideration for designing these experiments is the region or length of DNA to be excised by loxP recombination. While smaller regions ranging from a single exon to several exons can be flanked by loxP sites using a single plasmid-based donor repair template, flanking larger regions up to entire genes (10’s of kilobases) exceeds the capacity of a single plasmid donor, requiring two independent loxP insertion events from two independent donor repair templates.
Despite these challenges and considerations, efforts in the zebrafish community continue to yield further improvements on these methods, better facilitating the nuanced dissection of gene function in zebrafish and expanding the ever-growing genome editing toolkit available to the zebrafish community.
- Katsnelson, 2019: https://www.nature.com/articles/s41684-018-0229-y
- Burg et al., 2018: Burg, Leonard, et al. “Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish.” PLoS genetics 14.11 (2018): e1007754.
- Boel, Annekatrien, et al. “CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments.” Disease models & mechanisms 11.10 (2018): dmm035352.
- Hoshijima, Kazuyuki, Michael J. Jurynec, and David Jonah Grunwald. “Precise editing of the zebrafish genome made simple and efficient.” Developmental cell 36.6 (2016): 654-667.