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

Mark Andrew Moosburner,Vincent A Bielinski,Pardis Gholami,Andrew E Allen,James K Mccarthy,Maxine Tan

doi:10.3389/fmicb.2020.00005

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, bacterial-conjugation 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 single-gene 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.

Highlights

Marine diatoms are of biotechnological significance for their capacity to assimilate large amounts of carbon and nitrogen, divide rapidly (Cermeño et al, 2011), thrive at high cell densities, and fix carbon via photosynthesis
The cultures were grown on Luria-Bertani broth or agar and supplemented with the following antibiotics when necessary: ampicillin (100 mg/L), carbicillin (100 mg/L), tetracyclin (10 mg/L), gentamicin (20 mg/L), zeocin (25 mg/L)
Negative selection against RFP was used for cloning an individual sgRNA expression cassette where negative E. coli colonies were visibly red

Summary

Introduction

Marine diatoms are of biotechnological significance for their capacity to assimilate large amounts of carbon and nitrogen, divide rapidly (Cermeño et al, 2011), thrive at high cell densities, and fix carbon via photosynthesis. Diatoms shunt nutrients toward metabolic processes that produce industrially valuable products such as biodiesel precursors (triglycerides a.k.a. TAGs), polyunsaturated fatty acids (PUFAs), and photosynthetic pigments (Pulz and Gross, 2004; Vinayak et al, 2015). TAGs), polyunsaturated fatty acids (PUFAs), and photosynthetic pigments (Pulz and Gross, 2004; Vinayak et al, 2015) Regardless of their innate ability to produce these high-valued molecules (HVMs), Abbreviations: GA, Gibson assembly; GG, golden gate assembly; Phaeodactylum, Phaeodactylum tricornutum; RT, room temperature. Utilization of genetic engineering technologies could shift metabolic outputs of marine diatoms toward these HVMs and improve commercial production where algae have been suggested as biological platforms for industrial feedstock and nutraceutical production (Vinayak et al, 2015). The development and optimization of such genetic engineering technologies is necessary before they can be safely, and robustly employed

Methods

Results

Conclusion