Multiplexed Knockouts in the Model Diatom Phaeodactylum by Episomal Delivery of a Selectable Cas9

Abstract

Marine diatoms are eukaryotic microalgae that play significant ecological and biogeochemical roles in oceans. They also have significant potential as organismal platforms for exploitation to address biotechnological and industrial goals. In order to address both modes of research, sophisticated molecular and genetic tools are required. We presented here new and improved methodologies for introducing CRISPR-Cas9 to the model diatom Phaeodactylum tricornutum cells and a streamlined protocol for genotyping mutant cell lines with previously unknown phenotypes. First, bacterialconjugation was optimized for the delivery of Cas9 by transcriptionally fusing Cas9 to a selectable marker by the 2A peptide. An episome cloning strategy using both negative and positive selection was developed to streamline CRISPR-episome assembly. Next, cell line picking and genotyping strategies, that utilize manual sequencing curation, TIDE sequencing analysis, and a T7 endonuclease assay, were developed to shorten the time required to generate mutants. Following this new experimental pipeline, both singlegene and two-gene knockout cell lines were generated at mutagenesis efficiencies of 48% and 25%, respectively. Lastly, a protocol for precise gene insertions via CRISPR-Cas9 targeting was developed using particle-bombardment transformation methods. Overall, the novel Cas9 episome design and improved genotyping methods presented here allow for quick and easy genotyping and isolation of Phaeodactylum mutant cell lines (less than 3 weeks) without relying on a known phenotype to screen for mutants.

N- and O-Glycosylation Pathways in the Microalgae Polyphyletic Group

“…This represents an important challenge for the next decades. However, it would benefit from the recent development of genome-editing tools in microalgae (Daboussi et al, 2014;Mussgnug, 2015;Baek et al, 2016;Nymark et al, 2016;Shin et al, 2016;Wang et al, 2016;Huang and Daboussi, 2017;Dumontier et al, 2018;Slattery et al, 2018;Guzmán-Zapata et al, 2019;Fabris et al, 2020;Moosburner et al, 2020;Park et al, 2020). Moreover, several recent studies carried out in plants have highlighted the feasibility of N-and O-glycosylation metabolic engineering for the production of humanized recombinant N-and O-glycoproteins in transgenic plants including for example the production of recombinant IgA1 with defined human-type N-and O-linked glycans (Bakker et al, 2001;Paccalet et al, 2007;Vézina et al, 2009;Castilho et al, 2010;Castilho and Steinkellner, 2012;Yang et al, 2012;Dicker et al, 2016;Kallolimath et al, 2016).…”

Plasmid-based complementation of large deletions in Phaeodactylum tricornutum biosynthetic genes generated by Cas9 editing

“…In contrast, large deletions visible by long-read sequencing methodologies and our Nanopore data indicate that Cas9 and TevCas9 editing events result in deletions up to 2.7 kb in length. Cas9 editing with a single sgRNA in P. tricornutum could achieve the same goal as the paired Cas9 nickase strategy to specifically introduce large deletions 37 , and may be complementary with recently developed methods to multiplex sgRNAs on Cas9-editing plasmids for P. tricornutum 10 . Regardless, a better understanding of DNA repair pathways that operate on Cas9-introduced double-strand breaks will better inform strategies to bias repair events depending on the experimental goal.…”

Diatoms’ Breakthroughs in Biotechnology: <i>Phaeodactylum tricornutum</i> as a Model for Producing High-Added Value Molecules

“…Besides, in a recent work knockout of the diatom P. tricornutum has been established to create mutants by CRISPR-episome assembly. In this study, researchers developed new methodologies for the introduction of CRISPR/Cas9 in P. tricornutum cells and, other strategies for the rapid generation and isolation of CRISPR/Cas9 mutants [191]. Moreover, using diatom species in the fermentation approaches for the pilot-scale production seems to be promoting in many industrial fields such as antimicrobial production [133].…”