CRISPR/Cas9 knockin methodology for the sea urchin embryo

Abstract

CRISPR technology has revolutionized the biological research world, making animals heretofore recalcitrant to genetic manipulation, accessible to analysis of specific gene functions. Building upon the demonstration of targeted gene mutations in the sea urchin (CRISPR knockout) (Fleming et al., 2021; Lin et al., 2019; Lin & Su, 2016; Liu et al., 2019; Vyas et al., 2022), investigators may now be able to insert exogenous DNA into specific locations in the genome (CRISPR knockin). Such Cas9‐mediated knockins will reveal sites of gene expression, and function. By judicious selection of exogenously encoded tags, for example, a fluorescent reporter, an investigator may then follow specific gene activities and cell lineages throughout development in live embryos. This tag can also be used for protein pull‐down without requiring an antibody for the targeted protein. Here we describe a procedure for CRISPR‐based knock‐in DNA in the sea urchin Strongylocentrotus purpuratus. Sea urchin larvae produce echinochrome pigments that require several gene functions including the enzyme polyketide synthase 1 (PKS1) (Barsi et al., 2015; Calestani & Wessel, 2018; Calestani et al., 2003; Perillo et al., 2020; Wessel et al., 2020). Sp PKS1 expression is restricted to a small population of ∼50 cells of theVeg2 lineage of the animal (Barsi et al., 2015; Calestani et al., 2003). We realized that using PKS1 to evaluate CRISPR knockin success was highly stringent since the insertion must occur within that small lineage, and be expressed by yet a smaller population of the lineage. Mutations of the gene encoding PKS1 by CRISPR knockout resulted in albino larvae, an easy phenotype to assess with simple brightfield microscopy (Oulhen & Wessel, 2016a). A single gRNA was previously shown to mutate PKS1 by Cas9 activity, nearly 100% of the time in embryos from S. purpuratus and Hemicentrotus pulcherrimus (Liu et al., 2019; Oulhen & Wessel, 2016a; Oulhen et al., 2022). We took advantage of this highly efficient gRNA to test and to optimize Cas9‐ mediated methodology in the sea urchin S. purpuratus. We tested three different donor templates for their efficacy in selectively knocking‐in exogenous DNA encoding a fluorescent protein: plasmid DNA, linear double‐stranded DNA, single stranded DNA. The key for this test is a highly efficient gRNA against the target gene, and a DNA repair template that contains homologous regions to the target sequence for homology directed repair (Figure S1). Investigators have previously injected linear DNA into sea urchin eggs/early embryos, which results in rapid and extensive concatenation (McMahon et al., 1985) that appears to be detrimental to high‐fidelity insertion (data not shown). To counter this concern, we tested circular plasmid‐based strategies. Here, the DNA repair template targeting the cleaved genomic locus was contained within a plasmid and was accessible for insertion before or following CRISPR‐Cas9 cutting of the same flanking sequence in the plasmid as in the targeted genomic locus. This strategy resulted in GFP insertions into PKS1, but was inconsistent for reasons not yet clear (Figures S2 and S3). We obtained the best results using a different strategy based on a double strand PCR product as the DNA repair template whose termini were blocked from concatenation (Gutierrez‐Triana et al., 2018; Kimura et al., 2014; Paix et al., 2017, 2019; Seleit et al., 2021; Winkler et al., 1991). We provide a detailed protocol in the Supporting Information document explaining how to insert a Neon fluorescent tag in the gene Sp PKS1 using this strategy. This approach relies on a double‐stranded DNA donor (single stranded DNA did not yield insertions; Figure S1) containing short homology arms from the Sp PKS1 gene, flanking a fluorescent protein reporter sequence (Figures 1 and 2). The fluorescent protein Neon was selected for this