Evaluation of Alcalase pretreatment for Chlamydomonas reinhardtii CRISPR knock-in

Abstract

We wanted to use CRISPR-Cas9-mediated homology-directed repair (HDR) in C. reinhardtii to insert genomic landing pads as part of a larger project. In cell-walled algae, delivering Cas9 ribonucleoproteins (RNPs) and donor DNA requires first digesting that wall. The standard approach uses autolysin, a gamete-derived protease that must be prepared in-house over several days and varies in activity between batches. We tested whether Alcalase, a commercially available serine endopeptidase, could serve as an off-the-shelf alternative. We tested Alcalase pretreatment and compared it to GeneArt MAX Efficiency Transformation Reagent for Algae, which facilitates transformation and random integration of donor DNA. We electroporated Cas9 RNPs carrying one of four guide RNAs plus an antibiotic-resistance donor gene into our cells and found that Alcalase digested cell walls, but reduced viability independent of electroporation. Further, surviving cells were less likely to produce nourseothricin (NAT)-resistant colonies. Amplicon sequencing of NAT-resistant colonies detected no knock-in events or target-site indels with either method, indicating that on-target editing efficiency was low and NAT resistance arose primarily from off-target genomic integration of donor DNA. We stopped pursuing this work after bigger-picture priorities shifted, but are sharing our initial findings in case this is useful to others considering this approach. One attempt under one set of conditions isn't enough to rule out Alcalase pretreatment for targeted gene integration in Chlamydomonas, and we think this could be worth pursuing using gentler conditions.