Vicencio, Jeremy, Kukhtar, Dmytro, CERON, Julian, Ruiz, Miguel, Brena, David, Cots, Mariona
[
International Worm Meeting,
2021]
Many C. elegans CRISPR-based genome editing protocols have been developed in the last few years, ranging from plasmid-based approaches to cloning-free methods. We contributed to this collection of methods with nested CRISPR (Vicencio et al., Genetics 2019), facilitating the efficient and cloning-free generation of knock-in strains. We have also investigated the relationship between fragment length and efficiency to determine insertion limits given a single double-strand break. Our data demonstrate that insertion efficiencies decrease as the inserted fragment's length increases from 600 bp to 1600 bp. In terms of expanding our tools, we have developed new nested CRISPR sequences for the insertion of SL2::mCherry, GFP::H2B, and GFP::degron::3xFLAG tags. We are interested in testing the efficiency of nucleases aside from Cas9 to overcome the NGG PAM limitation. Our comparative studies of Cas9 and Cas12a (TTTV) PAM indicate that both nucleases are equally efficient for inserting a fluorescent tag. We have also explored the use of Cas9 variants with minimal PAM requirements, namely SpG (NGN) and SpRY (NRN > NYN). Our preliminary results using ribonucleoproteins (RNPs) and a strain endogenously producing SpG Cas9 validate their use in C. elegans, albeit with adjusted conditions, to produce both imprecise indels and precise knock-ins. As examples, we observe up to 80% efficiency for knocking out a wrmScarlet reporter and up to 20% efficiency for knocking in a 100-bp fragment using SpG (NGC PAM). Overall, these tools will further expand genome editing possibilities in C. elegans with new nucleases that allow editing of previously inaccessible sites and an expanded catalog of nested CRISPR sequences that require little to no additional optimization.