Rapid and precise genome editing in a marine diatom, Thalassiosira pseudonana by Cas9 nickase (D10A)

Abstract Diatoms are dominant phytoplankton accounting for up to 20% of annual global primary production. Diatoms are also one of the first model organisms amongst secondary endosymbionts, which have a highly chimeric genome structure accommodating genes from multiple endosymbiosis partners in their evolutionary history. Despite their ecological importance and evolutionary uniqueness, molecular-level studies have lagged behind other model organisms, hindered by difficulties in controlling their ploidy and homologous recombination system. Recently, genome editing tools, such as clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 and transcription activator-like effector nucleases (TALEN), have been established in some diatoms. However, the improvement of CRISPR/Cas9 precision technology in diatoms is still underway, and the experimental complexity of TALEN limits their application in diatom molecular genetics. Cas9 nickase, a mutated version of Cas9 with a paired-single guide RNAs (sgRNAs), was recently developed to introduce a precise double cut of target DNA, suppressing the off-target effects of native Cas9 nuclease. In this study, we developed the Cas9 nickase (D10A) system for genome editing of the marine diatom Thalassiosira pseudonana. Using this system, we successfully introduced mutations into a gene encoding a putative θ-type carbonic anhydrase, a recently discovered class of carbonic anhydrase widespread in Stramenopiles. The results showed that this system introduced precise and relatively short biallelic indels to the diatom genome with minimal off-target effects.