Development of CRISPR/Cas9 in Nannochloropsis and other algae toward understanding and manipulating energy allocation

Abstract

Nannochloropsis is a genus of eukaryotic microalgae that grows well in outdoor bioreactors and produces high yields of triacylglycerols (TAGs), which can be processed into biodiesel. In this work, we chemically characterize the storage carbohydrate of Nannochloropsis for the first time, and interrupt enzymes required for its biosynthesis to (1) understand their function and (2) interrogate whether this unused biomass component can be eliminated without significant impact on viability. To generate targeted gene knockouts, we developed CRISPR/Cas9 methods for Nannochloropsis and first interrupted nitrate reductase, a common target for genetic knockout because it is both inessential and easily scored. The method and validated chassis strain was then used to interrogate the beta-glucan synthase (BGS) and transglycosylase (TGS) enzymes believed to be responsible for the backbone polymerization and branching of the storage beta glucan, respectively. We identified no significant growth defects in our laboratory culturing conditions but did confirm that both genes were fundamental to synthesis of this beta glucan storage carbohydrate. The generated knockouts of either gene do not produce the elevated carbohydrate phenotype of wild-type cells in response to nitrogen deprivation. We also observed a non-bleaching phenotype in knockouts of BGS, where chlorophyll and carotenoid content remain elevated in mature cultures when wild-type cultures reduce their chlorophyll and carotenoid content. The design and diagnostic CRISPR/Cas9 methods developed for Nannochloropsis were then modified to transform a fast-growing high-light-, high-heat-, and high-salt-tolerant microalga Chlorella sp. strain CCMP252 with complexed Cas9-sgRNA to generate insertional knockouts of the nitrate reductase gene, demonstrating the broad applicability of the lessons learned.