One-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum by DNA-free genome editing

Abstract

Recently developed transgenic techniques to explore and exploit the metabolic potential of microalgae present several drawbacks associated with the delivery of exogenous DNA into the cells and its subsequent integration at random sites within the genome. Here, we report a highly efficient multiplex genome-editing method in the diatom Phaeodactylum tricornutum, relying on the biolistic delivery of CRISPR-Cas9 ribonucleoproteins coupled with the identification of two endogenous counter-selectable markers, PtUMPS and PtAPT. First, we demonstrate the functionality of RNP delivery by positively selecting the disruption of each of these genes. Then, we illustrate the potential of the approach for multiplexing by generating double-gene knock-out strains, with 65% to 100% efficiency, using RNPs targeting one of these markers and PtAureo1a, a photoreceptor-encoding gene. Finally, we created triple knock-out strains in one step by delivering six RNP complexes into Phaeodactylum cells. This approach could readily be applied to other hard-to-transfect organisms of biotechnological interest.

Cas9-generated auxotrophs ofPhaeodactylum tricornutumare characterized by small and large deletions that can be complemented by plasmid-based genes

“…Additionally, the knockouts survived on 5-FOA concentrations that fully inhibited the growth of wild-type P. tricornutum ( Figure 4A). This is consistent with phenotypes previously observed for P. tricornutum UMPS knockouts 7,21 . There was a slight growth advantage of ∆UMPS1 over ∆UMPS2 on media supplemented with both 5-FOA and uracil, but not on media containing uracil alone.…”

“…The available tools for genetic manipulation of P. tricornutum have grown substantially in recent years, including the adaptation of TALEN and Cas9 genome-editing nucleases for targeted knockouts [6][7][8][9]30 . Examination of edited sites in P. tricornutum and other organisms has revealed that many Cas9 events are not homogeneous.…”

“…A variety of plasmid-based genetic tools have been developed for P. tricornutum that facilitate basic molecular manipulations and expression of complex synthetic pathways [3][4][5][6] . We, and others, have developed plasmid-based and DNA-free CRISPR (clustered regularly interspaced palindromic repeats) reagents for targeted editing of the P. tricornutum chromosome using the Cas9 protein (CRISPR-associated protein 9) [6][7][8][9][10] . These plasmid-based tools and synthetic pathways are currently maintained by available antibiotic-based selections, including Zeocin TM , phleomycin, nourseothricin, and blasticidin-S and their respective resistance genes, Sh ble, nat, and bsr [11][12][13][14] .…”

The Synthetic Biology Toolkit for Photosynthetic Microorganisms

“…A recent breakthrough was the development of genome editing through Zn-Finger Nucleases, Transcription Activator-Like Effector Nucleases (TALENs), and CRISPR/Cas9 for different eukaryotic algae. TALENs were successfully used for P. tricornutum (Weyman et al, 2015;Serif et al, 2018), whereas Znfinger nucleases were developed for Chlamydomonas (Sizova et al, 2013;Greiner et al, 2017). CRISPR/Cas9 genome editing was achieved in Chlamydomonas (Shin et al, 2016;Greiner et al, 2017), P. tricornutum (Nymark et al, 2016;Serif et al, 2018;Slattery et al, 2018), and T. pseudonana (Hopes et al, 2016).…”

Potentiation of concurrent expression of lipogenic genes by novel strong promoters in the oleaginous microalga Phaeodactylum tricornutum

“…The eukaryotic microalga Phaeodactylum tricornutum is considered an attractive candidate for the production of a wide range commodity components (Kikutani et al, ; Xue et al, ). With the availability of sequenced genome and genetic toolkit, P. tricornutum has garnered huge research attention for metabolic engineering (Niu et al, ; Serif et al, ). Harnessing lipid metabolic pathways in oleaginous microalgae hold great potential for scalable biofuel production (Xue et al, ).…”