Enhancing CRISPR/Cas-Mediated Gene Knockout With Short Non-Homologous Oligonucleotides

Area of Science:

• Synthetic Biology
• Molecular Biology
• Algal Biotechnology

Background:

• Chlamydomonas reinhardtii is a model green alga with significant industrial potential for producing recombinant proteins and chemicals.
• CRISPR-Cas technology enables genome editing in C. reinhardtii, but low gene knockout (KO) efficiency limits its application in pathway engineering and functional genomics.

Purpose of the Study:

• To enhance the efficacy of CRISPR-Cas-mediated gene knockout in Chlamydomonas reinhardtii.
• To investigate the mechanism and influencing factors of enhanced gene knockout efficiency.

Main Methods:

• Co-delivery of CRISPR-Cas reagents with short double-stranded non-homologous oligodeoxynucleotides (dsNHOs).
• Analysis of dsNHO length, structure, and chemical modifications.
• Investigation of the role of KU70/80 (KU) heterodimer in the enhancement process.
• Assessment of gene knockout efficacy across different loci and strains.

Main Results:

• Co-delivery of dsNHOs increased gene knockout efficacy by up to 100-fold.
• The enhancement, termed non-homologous oligonucleotide enhancement (NOE), is dependent on dsNHO characteristics.
• NOE is mediated by the KU70/80 heterodimer in a manner independent of Cas nuclease, locus, or strain.
• dsNHOs appear to disrupt DNA double-stranded break sensing pathways, favoring microhomology-mediated end joining (MMEJ) over canonical non-homologous end joining (c-NHEJ).

Conclusions:

• Non-homologous oligonucleotide enhancement (NOE) is a potent strategy for significantly improving CRISPR-Cas-mediated gene knockout efficiency in Chlamydomonas reinhardtii.
• This method offers a valuable tool for advancing synthetic biology and functional genomic studies in this model alga.
• The findings suggest potential applications for NOE in other organisms for enhancing gene editing outcomes.