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

Daniel Guzmán-Zapata,Noé Durán-Figueroa,Fabien Nogué,Jesús Badillo-Corona,María Oliver-Salvador,José Castrejón-Flores,Edgar Salgado-Manjarrez,Karla Macedo-Osorio,José Sandoval-Vargas

doi:10.3390/ijms20051247

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.

Highlights

Since its development as a biotechnological tool and its first applications, CRISPR/CRISPR-associated protein 9 (Cas9) technology has been proposed as a simple and precise tool for genome editing [1,2,3]
To investigate whether transient expression of S. pyogenes Cas9 along with its cognate single guide RNA (sgRNA) could be used for genome editing in the photosynthetic alga C. reinhardtii, we chose to target the Adenine phosphoribosyl transferase (APT) gene
We reasoned that a Cas9-mediated double strand break (DSB) in the coding region of the adenine phosphoribosyl transferase (APT) gene in C. reinhardtii would result in loss of function in this gene, following insertions or deletions caused by the error-prone Non-homologous end joining/Alternative end joining (NHEJ/AltEJ) repair mechanisms [10]

Summary

Introduction

Since its development as a biotechnological tool and its first applications, CRISPR/Cas technology has been proposed as a simple and precise tool for genome editing [1,2,3]. It has been successfully established in organisms across all kingdoms of life, including bacteria [4], yeasts [5], plants [6], mice [7] and human cells [3,8]. In its most basic application, genome editing is achieved by constitutive or transient expression from vectors containing the Cas and sgRNA sequences in the cells of interest. Engineered forms of Cas and algorithm-mediated selection of non-redundant target sites make the technology more versatile, precise and reproducible than previously developed methods for genome editing, such as meganucleases, zinc fingers, and TALENs [15]

Methods

Results

Conclusion