Investigating CRISPR-mediated gene editing and its relationship with DNA repair in Chlamydomonas reinhardtii

Abstract

Chlamydomonas reinhardtii is a model green microalga that has great industrial potential to become the sustainable bio-factory for recombinant protein and high value chemicals production. Efficient genome editing tools are required to redesign this organism for synthetic biology applications. CRISPR-Cas editing technologies have already been adapted for gene knockout, transgene knock-in and precise gene editing in C. reinhardtii. However, low CRISPR/Cas-mediated gene knockout efficacy hampers its capacity for multiplex pathway engineering and functional genomic studies via simultaneous knockout of multiple genes.

We found that co-transfection of CRISPR-Cas gene editing reagents with short double-stranded non-homologous oligodeoxynucleotides (dsNHO) increased the gene knockout efficacy of the endogenous FKB12 locus by up to 100-fold in C. reinhardtii. This gene knockout boosting phenomenon was previously coined as non-homologous oligonucleotide enhancement (NOE) of gene editing. NOE is observed in other endogenous loci, in different C. reinhardtii strains, and works with both Cas9 and Cas12a (Cpf1) CRISPR proteins. By investigating the impact of length, structure, and chemical modifications of dsNHO on NOE, our result revealed that any secondary DNA structure with a minimum of 24 base pairs can induce NOE, and the dsNHO termini is important for NOE.

We next explored the underpinning genetic pathways and potential mechanisms of NOE using reverse genetics. We show evidence that KU70/80 heterodimer could be involved as it is a major DNA double-stranded break sensor in C. reinhardtii. By understanding the mechanism of NOE, we can manipulate proteins-of-interest directly with fast, targeted protein degradation technologies to boost knockout efficacy. Hence, the thesis additionally explores whether the auxin-inducible degron (AID) technology could be developed in C. reinhardtii, with future aims to manipulate DNA repair pathways for CRISPR editing. Finally, we report additional investigations into improving precise gene editing efficacy in C. reinhardtii using the single-strand templated repair (SSTR) technology, and into the role of DNA repair proteins involved. Our research into NOE as knockout editing boosters will hopefully help accelerate fundamental biology research in C. reinhardtii.