Efficient Editing of the Nuclear APT Reporter Gene in Chlamydomonas reinhardtii via Expression of a CRISPR-Cas9 Module

Abstract

The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a versatile and useful tool to perform genome editing in different organisms ranging from bacteria and yeast to plants and mammalian cells. For a couple of years, it was believed that the system was inefficient and toxic in the alga <i>Chlamydomonas reinhardtii</i>. However, recently the system has been successfully implemented in this model organism, albeit relying mostly on the electroporation of ribonucleoproteins (RNPs) into cell wall deficient strains. This requires a constant source of RNPs and limits the application of the technology to strains that are not necessarily the most relevant from a biotechnological point of view. Here, we show that transient expression of the <i>Streptococcus pyogenes</i> Cas9 gene and sgRNAs, targeted to the single-copy nuclear <i>apt9</i> gene, encoding an adenine phosphoribosyl transferase (<i>APT</i>), results in efficient disruption at the expected locus. Introduction of <i>indels</i> to the <i>apt9</i> locus results in cell insensitivity to the otherwise toxic compound 2-fluoroadenine (2-FA). We have used agitation with glass beads and particle bombardment to introduce the plasmids carrying the coding sequences for Cas9 and the sgRNAs in a cell-walled strain of <i>C. reinhardtii</i> (CC-125). Using sgRNAs targeting exons 1 and 3 of <i>apt9</i>, we obtained disruption efficiencies of 3 and 30% on preselected 2-FA resistant colonies, respectively. Our results show that transient expression of Cas9 and a sgRNA can be used for editing of the nuclear genome inexpensively and at high efficiency. Targeting of the <i>APT</i> gene could potentially be used as a pre-selection marker for multiplexed editing or disruption of genes of interest.