A fast and reliable strategy to generate TALEN-mediated gene knockouts in the diatom Phaeodactylum tricornutum

Abstract

Reverse genetics techniques are powerful tools for studying gene functions. In the model diatom Phaeodactylum tricornutum, RNAi-mediated knockdown of genes still is the most commonly used reverse genetics technique. Due to the diploidic life cycle missing reproduction in lab cultures, many commonly used techniques to create knockout instead of knockdown lines are not applicable in P. tricornutum. These limitations can be overcome by using genome editing approaches like TALEN (Transcription activator-like effector nucleases), and/or CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats), allowing the introduction of targeted mutagenesis events. Both techniques have recently been adapted exemplarily for diatoms, however, no concise guidelines exist yet for routine utilization of these tools and the subsequent characterization of the mutants. We therefore have adapted a cost-effective TALEN generation system previously established for mammalian cells for the use in P. tricornutum, allowing the assembly of TALENs in about two weeks. We further provide protocols for: a) choosing a TALEN target site in order to avoid potentially ineffective and/or off-target prone TALEN constructs, b) efficient transformation of P. tricornutum with both TALEN constructs, utilizing two antibiotics resistance markers, c) effective screening of the transformants. In order to test our system we chose the blue-light dependent transcription factor Aureochrome 1a (PtAureo1a) as a target gene due to the known phenotype of previously characterized P. tricornutum RNAi knockdown strains. Our TALEN approach appears to be highly efficient: targeted mutation events were detected in 50% of all transformants obtained, whereas 21% of the transformants were found to be bi-allelic knockout lines. Furthermore, most TALEN transformed cell lines were found to be genetically homogeneous without the need for re-plating, which greatly facilitates the screening process..

Genome editing in diatoms: achievements and goals

“…Technical improvements should decrease the TALENs production costs and increase the feasibility of producing these endonucleases in a short time. A protocol by Sanjana et al (2012) for generating fast, easy, and affordable TALEN constructs, based on a library of monomers, each representing one repeat unit of a TALE, and a two-step Golden-gate reaction, has recently been adopted for diatoms generating knockout strains for blue light Aureochrome photoreceptors in P. tricornutum (Serif et al 2017). Moreover, compact TALENs (cTALENs) (Beurdeley et al 2013), a new TALEN scaffold where the FokI catalytic head has been replaced by the I-TevI homing endonuclease, have been designed for diatoms.…”

“…For temporary induction or inhibition of endonuclease expression, the nitrate reductase (NR) promoter has been used, as the expression of the NR gene can be induced or inhibited by exchanging ammonia by nitrate as a nitrogen source or vice versa. The NR promoter construct, originally developed for the diatom Cylindrotheca fusiformis (Poulsen and Kröger 2005), and then adapted for P. tricornutum and T. pseudonana (Poulsen et al 2006;Miyagawa et al 2009;Hempel et al 2011;Chu et al 2016;Lau et al 2016), has already been used to drive a strong expression of TALEN (Serif et al 2017) and Cas9 (Stukenberg et al 2018) in P. tricornutum cells grown in nitrate-containing media. However, studies of NR promotor-driven GFP expression in P. tricornutum also indicated that gene expression cannot be completely switched off in the absence of nitrate (Chu et al 2016).…”

“…Multiple traces with overlapping peaks at the mutation sites will be obtained in case of heterozygous biallelic or monoallelic mutations. To differentiate mutations in the two alleles, allele-specific primers can be generated from genome-sequencing trace files (see Serif et al 2017).…”

“…Southern blots together with PCR/sequencing was also the method of choice for a TALEN-based homologous recombination approach by Weyman et al (2015), which was based on co-transformation of P. tricornutum with two plasmids, one containing both TALENs and a plasmid containing a homolog recombination fragment including a resistance cassette for selection of the cell lines. In nearly all diatom studies that were based on NHEJ repair mechanisms, large insertions have been observed in the resulting cell lines (Daboussi et al 2014;Nymark et al 2016;Serif et al 2017). These insertions mostly consist of vector fragments being inserted at the DSB site, which may be due to the use of the biolistic transformation methods that result in shearing of the plasmid DNA.…”

Efficient CRISPR/Cas-mediated homologous recombination in the model diatomThalassiosira pseudonana

“…Genome-enabled studies have revealed genes, regulatory elements and epigenetic factors potentially controlling the expression of diverse phenotypes. It is only recently, however, that genome editing tools such as CRISPR/Cas (Sander and Joung 2014;Doudna and Charpentier 2014;Lander 2016) and TALENs (Gaj, Gersbach, and Barbas 2013) have become available, including in diatoms (Daboussi et al 2014;Hopes et al 2016;Nymark et al 2016;Weyman et al 2015;Serif et al 2017), enabling us to improve our understanding of individual genetic factors for the evolution and biology of diatoms. The introduction of gene-targeting by high frequency HR allows precise genetic changes via knock-in of a particular sequence at a specific locus, rather than just gene knock-out.…”