Validated CRISPR/Cas9 guide RNAs targeting neurodevelopmental genes in the tunicate Ciona robusta.

The tunicate Ciona robusta provides a powerful and simplified model for dissecting the genetic control of developmental and cell biology. With a larval CNS composed of just over 200 neurons and sensory cells, it has also emerged as a model organism for neurobiology and the development of the nervous system. Although CRISPR/Cas9-mediated mutagenesis is now routinely used in Ciona as an important technique used to interrogate gene function in diverse biological processes, validated single-guide RNAs (sgRNAs) have yet to be validated for several key neural genes. Here, we report the design and experimental validation of 25 novel sgRNAs targeting eight conserved genes encoding conserved proteins involved in neurodevelopment and neural function, including six transcription factors (Cdx, Foxb, Sox1/2/3, Dmbx, Engrailed, and Mnx) and two neural effector genes (Tyrosinase and Slc18a3/VAChT). Candidate sgRNAs were selected and tested for mutagenesis efficiency using Illumina-based target site amplicon sequencing. All sgRNAs induced insertions or deletions at their target loci, with most genes yielding at least one sgRNA with mutagenesis efficacy exceeding 30%, with the exception of Dmbx, for which maximal efficacy reached 25%. We further compared measured mutagenesis rates to scores generated by different predictive algorithms, observing a modest but potentially improved correlation with predictions based on a newer algorithm. Based on these results, we recommend considering both scoring algorithms in combination, for improved predictive value for Ciona.