A gene-within-a-gene Cas9/sgRNA hybrid construct enables gene editing and gene replacement strategies in Chlamydomonas reinhardtii

Previous studies demonstrated highly inefficient gene editing in C. reinhardtii using conventional Cas9 and sgRNA genes (only 1 editing event using > 1.5 x 10(9) initial cells). Design and testing of a hybrid gene-within-agene construct (composed of a Cas9 gene containing an artificial intron with an inserted sgRNA gene) demonstrated that such constructs were functional both in tobacco cells and C. reinhardtii cells. In tests with C. reinhardtii, approximately one in every similar to 3 x 10(7) initial cells contained an edited version of the targeted FKB12 gene (i.e., an average of similar to 3 colonies with an edited FKB12 gene per electroporation using 10(8) initial cells). Lack of an intact Cas9/intron-sgRNA gene in cells carrying either of two different edited genes strongly suggested that editing was due to transient expression of the Cas9/intron-sgRNA gene and the likely toxicity of long-term expression of Cas9 in C. reinhardtii cells. Co-transformation of the arginine-requiring mutant, arg7-8, with Cas9/intron-sgRNA constructs and appropriately designed synthetic, 80 nucleotide ssDNAs complementary to the argininosuccinate lyase (ARG) gene led to successful homologous recombination or nucleotide replacement and production of arginine prototrophs. As a practical application, a similar ssDNA oligonucleotide targeting the acetolactate synthase (ALS) gene and an appropriate Cas9/intron-sgRNA construct was used to create cells resistant to the herbicide, sulfometuron methyl.