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 Chlamydomonas reinhardtii. 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 Streptococcus pyogenes Cas9 gene and sgRNAs, targeted to the single-copy nuclear apt9 gene, encoding an adenine phosphoribosyl transferase (APT), results in efficient disruption at the expected locus. Introduction of indels to the apt9 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 C. reinhardtii (CC-125). Using sgRNAs targeting exons 1 and 3 of apt9, 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 APT gene could potentially be used as a pre-selection marker for multiplexed editing or disruption of genes of interest.

Site-Specific Gene Knock-Out and On-Site Heterologous Gene Overexpression in Chlamydomonas reinhardtii via a CRISPR-Cas9-Mediated Knock-in Method

“…Recently, some reports suggested that gene selection can be achieved with high yields (up to 30%) through counter selection without the use of antibiotic genes (Jiang and Weeks, 2017;Serif et al, 2018;Guzmán-Zapata et al, 2019). However, these methods are mostly functional for specific genes and cannot be applied universally.…”

“…crRNA and tracrRNA can be physically linked to form single guide RNA (gRNA) (Jinek et al, 2012). In C. reinhardtii, the CRISPR-Cas9 was first applied via DNA vector system in 2014 and recently ribonucleoprotein (RNP) system has been developed (Jiang et al, 2014;Baek et al, 2016;Guzmán-Zapata et al, 2019). CRISPR-Cas9 system is the ideal tool for gene-editing; however, it requires efficient selective markers for reducing the time and labor.…”

“…Presently, phenotypic changes such as visual changes are used to improve the selection efficiency, however, the method is less efficient, requires a lot of labor (Baek et al, 2016;Shin et al, 2016;Greiner et al, 2017;Jeong et al, 2017b), and is not suitable for most genes. Therefore, gene-editing methods using counter-selective markers such as adenine phosphoribosyl transferase (APT), nitrate reductase (NR), peptidylprolyl isomerase (FKB12), tryptophan synthase beta subunit (TSB), and orotidine 5 -phosphate decarboxylase (UMP) have been recently proposed (Shin et al, 2016;Wang et al, 2016;Jiang and Weeks, 2017;Serif et al, 2018;Guzmán-Zapata et al, 2019). Counter selection can be efficient; however, they are eventually dependent on specific phenotypes.…”

TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii

“…This raised concerns that Cas9 was toxic in C. reinhardtii. More recent studies, exploring different approaches, have clearly demonstrated that CRISPR/Cas9 is useful for targeted disruption of genes in C. reinhardtii [15][16][17][18][19][20][21][22]. However, some of the published methods have low efficiencies of achieving the desired edit, making it laborious to identify cells in which a gene has been successfully targeted, especially in the absence of a selectable marker.…”

Emerging Technologies in Algal Biotechnology: Toward the Establishment of a Sustainable, Algae-Based Bioeconomy

“…Although technology for precision genome editing, including zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), or CRISPR/Cas9, has been reported in many microalgae (Sizova et al, 2013;Weyman et al, 2015;Li et al, 2016;Nymark et al, 2016;Ajjawi et al, 2017), several challenges related to targeting, efficiency, and toxicity remain to be fully overcome. Strategies to circumvent these issues include transient Cas9 expression (Guzmán-Zapata et al, 2019), direct ribonucleoprotein (RNP) delivery (Baek et al, 2016;Shin et al, 2016) and use of Cas variants (Ungerer and Pakrasi, 2016). Marker-free and multiplex gene knock-out remains a challenge in some microalgae, although the use of multiple sgRNAs to multiplex genome editing targets has been shown to be feasible in diatoms (Serif et al, 2018).…”