Episomal tools for RNAi in the diatom Phaeodactylum tricornutum

Abstract

Background. The diatom Phaeodactylum tricornutum is a model photosynthetic organism. Functional genomic work in this organism has established a variety of genetic tools including RNA interference (RNAi). RNAi is a post-transcriptional regulatory process that can be utilized 25 to knockdown expression of genes of interest in eukaryotes. RNAi has been previously demonstrated in P. tricornutum, but in practice the efficiency of inducing RNAi is low. Methods.We developed an efficient method for construction of inverted repeat hairpins based on Golden Gate DNA assembly into a Gateway entry vector. The hairpin constructs were then 30 transferred to a variety of destination vectors through the Gateway recombination system. After recombining the hairpin into the destination vector, the resulting expression vector was mobilized into P. tricornutum using direct conjugation from E. coli. Because the hairpin expression vectors had sequences allowing for episomal maintenance in P. tricornutum, we tested whether a consistent, episomal location for hairpin expression improved RNAi induction Results. We successfully demonstrated that RNAi could be induced using hairpin constructs expressed from an episome. After testing two different reporter targets and a variety of hairpin sequences with 3 polymerase II and 2 polymerase III promoters, we achieved a maximal RNAi 40 induction efficiency of 25% of lines displaying knockdown of reporter activity by 50% or more. We created many useful genetic tools through this work including Gateway destination vectors for P. tricornutum expression from a variety of polymerase II and III promoters including the P. tricornutum FCPB, H4, and 49202 polymerase II promoters as well as the U6 and snRNA polymerase III promoters. We also created Gateway destination vectors that allow a cassette 45 cloned in an entry vector to be easily recombined into a transcriptional fusion with either ShBle or, for polymerase III promoters, the green fluorescent Spinach aptamer. Such transcriptional fusions allow for linkage of expression with a marker such as bleomycin resistance or fluorescence from the Spinach aptamer to easily select or screen for lines that maintain transgene expression. 50Discussion. While RNAi can be used as an effective tool for P. tricornutum genetics, especially where targeted knockouts may be lethal to the cell, induction of this process remains low efficiency. Techniques resulting in higher efficiency establishment of RNAi would be of great use to the diatom genetics community and would enable this technique to be used as a forward

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

“…For Golden Gate cloning, the sRNAi terminator was shortened by PCR to 112 bp to eliminate an internal BsaI restriction site. The sRNAi promoter and terminator was cloned from Phaeodactylum genomic DNA at the locus of the most highly expressed small, non-coding, intergenic RNA called sRNAi (Rogato et al, 2014;Bielinski et al, 2017). The sequences for the sRNAi promoter and terminator can be found in the Supplementary Material.…”

Multiplexed Genome Editing via an RNA Polymerase II Promoter-Driven sgRNA Array in the Diatom Phaeodactylum tricornutum: Insights Into the Role of StLDP

“…To date, the study of diatom molecular physiology has been dominated by Phaeodactylum tricornutum , having a fully sequenced and annotated genome ( Bowler et al, 2008 ) and a growing genetic manipulation toolkit ( Apt et al, 1996 ; Zaslavskaia et al, 2000 ; Karas et al, 2015 ; Diner et al, 2016 ; Bielinski et al, 2017 ; Serif et al, 2017 ; Buck et al, 2018 ; Taparia et al, 2019 ; Pollak et al, 2020 ). Moreover, P. tricornutum has been used for developing several biotechnological products via either heterologous expression ( Hempel et al, 2011 ) or the introduction of entire biosynthetic pathways using synthetic biology tools.…”

“…CRISPR/Cas9 technology is especially valuable for asexual species, such as the model diatom P. tricornutum , for which some of the tools used in forward or reverse genetics cannot be applied. Moreover, RNAi for gene expression knock-down is known to be unstable in this diatom ( Bielinski et al, 2017 ). The discovery of E. coli -mediated conjugation for the delivery of non-integrating extrachromosomal DNA (diatom episome) to the nucleus of P. tricornutum ( Karas et al, 2015 ; Diner et al, 2016 ) presented an ideal system for delivering CRISPR/Cas9 for genome editing ( Sharma et al, 2018 ; Slattery et al, 2018 ; Moosburner et al, 2020 ) without its integration into the nuclear genome.…”

Validating a Promoter Library for Application in Plasmid-Based Diatom Genetic Engineering

“…The GUS coding region and the fcpA terminator fragment were amplified from vectors PB-fcpB (previously PtRNAi-2c) and PtRNAi-3 . Two other fragments were also amplified from the vector PtRNAi-3.…”

“…The GUS coding region and the fcpA terminator fragment were amplified from vectors PB-fcpB (previously PtRNAi-2c) and PtRNAi-3. 58 Two other fragments were also amplified from the vector PtRNAi-3. One containing the ampR gene through the oriT region, whereas the other contained the tetR gene, including the CEN6-ARSH4-HIS3 region, to the beginning of the ShBle cassette (driven by P. tricornutum P fcpF ).…”