A fast and reliable strategy to generate TALEN-mediated gene knockouts in the diatom Phaeodactylum tricornutum
TL;DR: A cost-effective TALEN generation system previously established for mammalian cells for the use in P. tricornutum 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.
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.
Summary (3 min read)
- Diatoms are unicellular microalgae belonging to the Stramenopiles.
- The mandatory binding of both TALENs strongly increases the targeting specificity .
- While NHEJ occurs during the whole cell cycle, HR is mainly restricted to the late S and G2 phase .
2.1. Assembly of the TALEN plasmids
- The correct integration of the TALEN backbone was verified by Sanger sequencing (GATC, Konstanz, Germany).
- Cloning and insertion of the target sequences into their TALEN plasmids were performed as described in .
2.2. Cultivation of algae
- The P. tricornutum strain UTEX646 was obtained from the culture collection of algae of the University of Texas (UTEX, Austin, USA).
- P. tricornutumwasgrown axenically in liquid F/2mediumwithout added silica and 16.5‰ salt content or on solid F/2 media which contained additionally 1.2% (w/v) Bacto Agar (BD, Sparks, MD, USA).
- Plated cultureswere cultivated under continuous illumination at 75 μmol photons m−2 s−1 (Osram Biolux L30W/965).
2.3. Nuclear transformation of P. tricornutum
- Nuclear transformation of P. tricornutumwas performed using a BioRad Biolistic PDS-1000/He Particle Delivery System (Bio-Rad, Hercules, CA, USA) fitted with 900/1100/1350 psi rupture disks as described previously [13,38,39].
- For selective cultivation of P. tricornutum transformants, 75 mg mL−1 Zeocin (Invitrogen, Carlsbad, CA, USA) and 150 mg mL−1 Nourseothricin (ClonNat, Werner Bioagents, Jena, Germany) were added to the solid F/2 media [13,38].
2.4. DNA isolation
- Genomic DNA was isolated using the nexttec™ 1step DNA isolation from tissues & cells kit (Biozym, Hessisch Oldendorf, Germany) according to the manufacturer's instructions.
- Concentration of genomic DNAwasmeasured by Nanodrop 2000 UV/VIS Spectrometer (Thermo Fisher, Schwerte, Germany).
- 5. Allele-specific PCR DNA sequences for the two alleles of the PtAureo1a gene (JGI ID: 49,116) were deduced from the whole genome shotgun sequencing (WGS) database (NCBI) by alignment of the individual sequence reads.
- Allele-specific primers for PCR were derived which include an allele-specific difference on the 3′ terminal base, thereby preventing polymerases without proofreading function from amplifying the respective other allele.
- PCR was performed using either Taq B polymerase (Biozym, Hessisch Oldenburg, Germany) or HiDi polymerase (myPOLS, Konstanz, Germany) according to the manufacturer's instructions.
2.6. Southern blotting
- Isolated genomic DNA was digested using each of the following restriction enzymes overnight according to the manufacturer's instructions: BamHI, BsrGI, HindIII (Thermo Fisher, Schwerte, Germany).
- The agarose gel was incubated for 10 min in denaturation solution (0.5 M NaOH, 1 M NaCl), followed by 10 min incubation in neutralization solution (0.5M Tris-HCl, 3MNaCl, pH7.5).
- The blotting setup was then weighed down.
- The 400 bp DIGlabeled probe was synthesized using the PCR DIG Probe Synthesis Kit (Roche, 11636090910) using an PtAureo1a-containing plasmid  as template and primers Aureo1a_probe_for/rev (see Table SI).
2.7. Protein isolation and immunoblotting
- For protein extraction, cell pellets were resuspended in lysis buffer (4 M urea, 1.5 M thiourea, 1% SDS, 20 mM Tris pH 8) supplemented with protease inhibitor (Complete™ EDTA-free, Roche) according to the manufacturer's instructions.
- A spatula tip of 1 mm, 0.5 mm and 0.1 mm diameter beads was added and the cells were lysed in a Savant FastPrep FP120 bead mill (Thermo Scientific, Karlsruhe, Germany) six times for 20 s with cooling on ice for 1 min between each cycle.
- Each lane was loaded with protein extract of either wild type or mutant cell lines.
- After blotting, the nitrocellulose membrane (Amersham Protran 0.1 μm NC, GE Healthcare) was cut between 35 and 40 kDa and the top half (40–250 kDa) was used to detect PtAUREO1a, whereas the bottom half (0–35 kDa) was used to detect theD1 loading control.
- Blots were developed using an Odyssey FC Imaging System (Li-Cor, Bad Homburg, Germany).
2.9. Pigment extraction
- Extraction of pigments and subsequent analyses via HPLCwere done as described in .
- Samples were analyzed on a calibrated Hitachi LaChrom Elite HPLC system equipped with a Nucleosil 120-5 C18 column (Macherey-Nagel, Düren, Germany).
3.1. Generation of the TALEN constructs
- In order to perform a cost-effective and easy assembly of the TALEN targeting sequence, the authors chose a system developed previously for mammalian systems, allowing the complete assembly and sequence verification of individual TALEN plasmids within two weeks .
- When using two different antibiotic resistances on the two plasmids encoding the individual TALENs, a higher selection stringency is achieved by screening for strains that have integrated both plasmids.
- The recommended workflow for designing and generating TALEN constructs is presented in Fig. TALE-NT 2.0 was used to predict target sites in the gene of interest and potential off-targets for each TALEN pair based on the P. tricornutum RefSeq sequence (GCF_000150955.2).
- According to the SAPTA guidelines, composite scores above 30 are recommended for a high rate of genemodifications.
3.2. Screening of the obtained transformants and statistical evaluation
- While several allele-specific differences were identified (based on the sequenced P. tricornutum strain Pt1 (CCAP 1055/1)), the two alleles could not be amplified separately from Pt4 (UTEX 646), the strain used in this study.
- The authors could solve the problem by identifying a mixed trace peak (T/G) at position 81 of the PtAureo1a gene in the Pt4 wild type cells as well as in the mutants, which allowed distinguishing both alleles.
- For each of the chosen restriction enzymes, Southern Blots of genomic DNA of wild type strains showed a single band in the range of 3 to 5 kbp (see Fig. 4).
- While the shifted band of strain 14 seems to indicate a shorter fragment, the 143 bp insertion detected by PCR (see above) introduced a new HindIII site, thereby causing a shorter fragment size.
- As only the combination of sequencing of PCR-amplified target genes and Southern Blots using a target gene specific probe allowed us to identify all mutated strains, the authors highly recommend screening TALEN-transformants using both methods whenever possible.
3.3. Homogeneity of the generated mutants
- One mono-allelic knockout line (14) and four bi-allelic knockout lines (6, 8, 9 and 11) were spread onto individual agar plates, and three colonies of each line were re-isolated from single cell colonies.
- Southern Blots and allele-specific PCRs were repeated in order to prove that the reisolated clones were genetically identical regarding the mutated target site.
- The reason why previously published systems [28,30] did not obtain genetically homogeneous transformants, despite replating the cells onto selection media after 48 h, is unclear.
- Additionally, TALEN seems less prone to mismatched base pairing indicated by a reduced off-target frequency relative to targeted mutagenesis events compared to CRISPR using a randomized DNA library [54,55], as has been summarized in .
3.4. Phenotypic characterization of PtAUREO1a knockout mutants
- Previously characterized PtAUREO1a knockdown strains generated via RNAi showed a significantly reduced chlorophyll a (Chl a) content per cell compared to thewild type strain undermedium light conditions (Qphar 30; 30 μmol photons m−2 s−1 absorbed photosynthetic radiation) .
- Interestingly, the NPQ capacity was reduced by about 40% in the three knockout lines 6, 8 and 9 despite the increased amount of XC pigments, whereas cell line 11 expressing a truncated PtAUREO1a showed wild typeNPQ levels (See Table 2; Student's t-test, p b 0.001).
- These results highlight that in certain cases stable knockout lines may not only amplify phenotypic effects compared to silencing lines, butmay also provide completely different results.
- When approximating the volume of fusiform P. tricornutum, only the bi-allelic knockout lines showed significant decreases in cell volume compared to wild type cells.
- Such a reduced cell length and volume had not been observed for the previously characterized PtAUREO1a silencing strains , confirming that knockout approaches may yield much more obvious phenotypes than RNAi-based silencing.
- An efficient TALEN system has been established for the diatom P. tricornutum, which leads to a high frequency of targeted mutation events and yields mainly genetically homogeneous cell lines.
- Additionally, all important steps, from target site design, TALEN construction using an easy-to-follow and publically available modular construction system, up to screening of the mutants, have been addressed to avoid the potential pitfalls for knockout generation in P. tricornutum, like potential off-targets and inefficient TALEN proteins.
- The TALEN system presented here may expand the application of reverse genetics approaches for creation of P. tricornutum knockout mutants to a broader scientific community.
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