| Description |
CRISPR-Cas based cell lineage tracing relies on inducing combinations of mutations in developing embryos that can describe patterns of cell divisions in the embryo. While these tools promise to change our understanding of cell fate in developing embryos, current tools only generate mutations early in development. Therefore, later cell divisions are not recorded. GESTALT, one of the first CRISPR lineage tools, utilizes the type II CRISPR-Cas system from Streptococcus pyogenes (SpCas9) due to its high efficiency in cells and organisms. Several other CRISPR-Cas systems have been characterized in cells, but remain underutilized for cell lineage tracing. These systems provide an opportunity for multiplexed editing, increasing the amount of cell lineage information captured in GESTALT and allowing for parallel recording of other transient events in cells. We hypothesized that CRISPR-Cas systems originating from different bacterial species could operate independently in the same embryo, a feature we termed orthogonal This is because each system has a distinct single-guide RNA (sgRNA) or CRISPR-RNA (crRNA), and a specific protospacer adjacent motif (PAM) in their DNA target site. Additionally, we hypothesized that CRISPR-Cas activity in zebrafish could be regulated through co-expression of anti-CRISPR (Acr) proteins, offering the opportunity to control the timing of genome editing. Here, we describe a simple mutagenesis screen, and confirm that CRISPR-Cas systems from Streptococcus pyogenes (SpCas9), Streptococcus aureus (SaCas9), and Lachnospiraceae bacterium (LbCas12a, previously known as LbCpf1) are highly effective in zebrafish and are fully orthogonal systems. With this information we have developed new GESTALT barcodes for cell lineage tracing, containing arrays of target sites for SaCas9, LbCas12a, and a hybrid barcode containing target sites for all three systems. We also demonstrated that type II Acrs are effective inhibitors of SpCas9 and SaCas9 in zebrafish. These additional CRISPR-Cas systems and Acr proteins broaden the toolset for genome modification in zebrafish, further develop GESTALT, and enable combinatorial and intersectional strategies for spatiotemporal control of genome editing in zebrafish. |
