Knock-down of a ligIV homolog enables DNA integration via

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Knock-down of a ligIV homolog enables DNA integration via homologous recombination in the marine diatom Phaeodactylum tricornutum Max Angstenberger, Julia Krischer, Ozan Aktas, and Claudia Büchel ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00234 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 16, 2018

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ACS Synthetic Biology

Title: Knock-down of a ligIV homolog enables DNA integration via homologous recombination in the marine diatom Phaeodactylum tricornutum

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Authors: Max Angstenberger, Julia Krischer, Ozan Aktaş and Claudia Büchel*

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Institute of Molecular Biosciences, Goethe University Frankfurt, Max-von-LaueStraße 9, Biozentrum, 60438 Frankfurt am Main, Germany

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*Corresponding author: Tel.: +49 69 798 29602; fax: +49 69 798 29600 E-mail:

[email protected] (C. Büchel)

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Abstract:

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Genetic engineering of Phaeodactylum tricornutum as a model organism for diatoms

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is the basis of molecular and biochemical research, and can also be used in biotechnological approaches. So far, integration of foreign DNA into the genome happens randomly by non-homologous end joining (NHEJ), if the classical method of particle bombardment is used, with the danger of negative physiological side effects. Here we show that a putative gene for a DNA ligase IV homolog (ligIV) in P. tricornutum codes for a functional LigIV. The knock-down of ligIV in P. tricornutum via

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antisense RNA drastically enhances homologous recombination (HR) by interfering with the NHEJ pathway at its central DNA ligation step done by LigIV. This enables a specific integration of DNA at desired locations, greatly enhanced transformation rates and provides a new way of specifically altering the genome of P. tricornutum.

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Keywords: Phaeodactylum tricornutum, DNA ligase IV, knock-down, nonhomologous end joining, homologous recombination, gene targeting

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Introduction

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P. tricornutum is a unicellular eukaryotic alga that has become a model organism to

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study different molecular and biochemical aspects in diatoms and it is also used for biotechnological approaches with a broad range of possible applications1,2. Both basic research and biotechnology profit enormously from genetic accessibility given in P. tricornutum since its genome is completely sequenced3, EST (expressed

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sequence tag) data for analysis of gene expression are available4,5 and transformation protocols are established6.

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The standard biolistic transformation of P. tricornutum is based on unspecific integration of DNA into the genome and limits genetic transformations to overexpression of genes6 and RNA silencing studies7. During the transformation process, the foreign plasmid DNA is thought to break (randomly) leading to complete or incomplete integration by non-homologous end joining (NHEJ) at random locations. Negative physiological side effects are possible and low transformation rates are often observed, which might result from e.g. disruption of genes. Thus, e.g. in case of RNA silencing, usually many clones have to be compared in order to pinpoint the specific effects of the silencing, since it is otherwise difficult to relate phenotypical interpretations solely to the effects of integrated constructs.

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Newer developments for genetic engineering in P. tricornutum are targeted gene

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disruption by mega nucleases and transcription activator-like effector nucleases (TALENs8), as well as clustered regularly interspaced short palindromic repeats (CRISPR/Cas99). All three approaches can target genes specifically leading to gene knock-outs by nucleases. But in case of stable transformation by biolistic methods, they also depend mainly on the NHEJ pathway to integrate the nuclease DNA itself as well as targeting elements at random locations into the genome. Only if the nucleases are located on a plasmid and not permanently incorporated in the genome, i.e. by an episomal strategy, this can be avoided so far10.

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The nuclease mediated cut in a target sequence can be repaired either by NHEJ or homologous recombination (HR). The latter can be used to integrate homologous DNA at the nuclease mediated restriction sites, as e.g. shown by 11 for an urease gene using TALEN. But still additional unspecific integrations via NHEJ cannot always be excluded. Another problem are off-target restrictions by nucleases, as reported for CRISPR/Cas912 at least for human cells, which might also lead to physiological problems caused by e.g. gene disruption. For P. tricornutum off-target restrictions seem to be unlikely but cannot be safely excluded yet13.



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In P. tricornutum both HR and NHEJ pathways are present with numerous predicted

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genes (KEGG database: http://www.genome.jp/kegg-bin/show_pathway?pti03450, pti03440). Various proteins are active in the NHEJ complex (e.g. XRCC4, KU70/KU80, BRCT) in which the last step is mediated by a DNA ligase of type IV (LigIV), which ligates the non-homologous DNA ends. In other organisms (e.g. Neurospora crassa14, Pichia ciferrii15), it could be shown that the knock-out of a DNA ligase IV homolog and therefore inactivation of the NHEJ pathway led to a dramatic increase of HR as the preferred DNA uptake mechanism. This enabled a high gene targeting efficiency without side effects due to the avoidance of unspecific DNA integrational events. Since other proteins than LigIV of the NHEJ complex were reported to have additional functions beside their role in NHEJ (e.g. telomere regulation by KU8616), LigIV was chosen as the best target for specific NHEJ interference and consequent HR upregulation in P. tricornutum.

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In P. tricornutum, two identical gene copies of a LigIV homolog (ligIV) are annotated

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on different chromosomes (7 and 28). The aim of this work was to create otherwise physiologically intact ligIV knock-down lines in order to disturb the NHEJ pathway

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and up-regulate the HR pathway to integrate DNA at desired locations without additional unspecific events. To this end, the characterisation of the putative LigIV had to be carried out first to confirm its physiological role. Then, knock-down mutants were created and tested for the ratio of transformation events due to NHEJ or HR, as well as for the specifity of HR using single or multiple targets. Besides the proof of principle that HR is occurring with reasonable rate, the differences in length of the homologous regions used should reveal specific preferences of HR in P. tricornutum.

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Results and Discussion

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LigIV model of P. tricornutum

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In order to strengthen the assumption that the two identical gene copies of ligIV (Fig. 1) code for a DNA ligase of type IV, the coding sequence was modelled on the known structure of the human DNA ligase IV by phyre217 software (Fig. 2A-D). Though sequence identity is relatively low (29%), alignment confidence (Fig. 2A) and quality assessment (Fig. 2B) are quite good with an overall confidence of 100%. Especially the DNA ligase typical motifs of the DNA binding site (Fig. 2C, marked in red) and ATP binding site (Fig. 2D, marked in red) are well predicted, indicating a very likely function of LigIV as a DNA ligase of type IV.

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DNA ligation assay using recombinant LigIV_his

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To verify that LigIV from P. tricornutum functions as a DNA ligase, the capability of LigIV to ligate DNA was tested. Recombinant LigIV with an N-terminal 10xHis-tag

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(LigIV_his) and its corresponding empty vector (EV) control were expressed in E. coli

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and purified by Ni-NTA. A protein of the expected size (about 150 kDa) could be detected by immunoblotting using an antibody against the His-tag (Fig. 2E). In a DNA ligation assay (Fig. 2F), vector DNA linearized by restriction in the multiple cloning site was incubated with purified LigIV_his, EV eluate, H2O or T4 DNA ligase for up to one hour, and after heat inactivation subsequently used to transform E. coli. Counting of clones revealed that linearized vector DNA could be indeed only religated using LigIV_his, but not with the EV eluate or water. The use of T4 DNA ligase as a positive control led to an uncountable amount of clones. This result further supports the idea of LigIV of P. tricornutum as a true DNA ligase.

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Generation and characterization of ligIV knock-down mutants

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To decrease the mRNA level of ligIV, a fragment of the ligIV coding sequence (see

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Fig. 1) was amplified and cloned inversely into the expression vector pPha-T1 leading to the antisense construct pPha-T1_AS (Fig. 3A, S2). P. tricornutum wild type

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was transformed using pPha-T1_AS and resulted in an expectedly low transformation

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rate of 8 · 10-9 clones · 1 µg-1 DNA · 108 cells-1.

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Clones NAS3 and NAS4 were chosen for further work because of their decreased mRNA level of ligIV to about 50% in NAS3 and to 35% in NAS4 (Fig. 3B). The physiological tolerance to the ligIV knock-down under normal conditions in NAS3 and

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NAS4 was confirmed by an unchanged PSII quantum yield (Fig. 3C) and a similar growth behaviour compared to wild type under ordinary low light as well as high light conditions (see suppl. Fig. S3). Since a ligIV knock-down was expected to increase the sensitivity of the cells against DNA damage, NAS3 and NAS4 were tested for their sensitivity against UV radiation (Fig. 3D). They indeed showed an UV radiation sensitive phenotype, in accordance with the assumption that ligIV codes for a DNA ligase of type IV in P. tricornutum. NAS3 and NAS4 were tested regularly for the expression of the knock-down construct and their growth rates. Even after 3 years no changes could be observed (data not shown).

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Integration of DNA in wild type

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DNA integration in wild type was reported to be unspecific, i.e. constructs insert randomly into the genome by NHEJ, as seen in6. This was also the case for the antisense constructs of the ligIV knock-down lines NAS3 and NAS4, shown by southern blot (Fig. 4A) when probed against ble, i.e. the antibiotic resistance conferred by transformation (see suppl. Fig. S2). In both knock-down lines, several different fragments show that insertions of the antisense construct took place at different genomic locations. In NAS3 at least two, and in NAS4 at least seven

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integrational events took place. These were not due to HR, which might have occurred since the antisense construct had an FcpA promotor and terminator. Testing for HR using different restriction enzymes for southern blots and probing with fcpA, a probe against the 5’ region of fcpA, for none of the fragments size changes compared to WT or consistent congruency to ble was detected (data not shown).

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The dominance of NHEJ as the preferred DNA integration mechanism could be further supported when wild type was transformed with a vector constructed for HR on target ligIV, called HRL and clones were analysed by southern blot (Fig. 4B). This vector contained a nat-cassette with fcpA promotor/terminator, surrounded by 1 kb flanking regions homologous to the ligIV gene (see Fig. 5A). The transformation rate

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was again low with 3 · 10-9 clones · 1 µg-1 DNA · 108 cells-1 and random insertions

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could be observed due to different fragment patterns in different clones on a southern blot (Fig. 4B) when probed against nat, the antibiotic resistance included in the HRL construct. Thus, transformation of wild type led to NHEJ driven unspecific DNA integration, independent of the construct used.

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The low transformation efficiency implies that DNA insertion via NHEJ itself is quite inefficient and/or many transformed cells die due to negative physiological side effects of unspecific integrations. Like often observed, many of the clones from wild type transformations showed impaired growth behaviour, which can also be explained by side effects.

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Integration of HRL in ligIV knock-down lines

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In contrast to wild type, transformation of the ligIV knock-down lines NAS3 and NAS4

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with construct HRL led to dramatically increased transformation rates of 7.5 · 10-6

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clones · 1 µg-1 DNA · 108 cells-1 for NAS3 and 1.2 · 10-7 clones · 1 µg-1 DNA · 108

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cells-1 for NAS4. The rate was even about two times (NAS4) to four times (NAS3) higher when linearised DNA was used for transformation. The higher transformation efficiency of NAS3 compared to NAS4 cannot be explained by the remaining ligIV

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expression, since NAS4 has a stronger knock-down than NAS3 in accordance with a higher occurrence of integrated antisense constructs. It therefore must have another reason related to the integration site(s) of antisense constructs in the genome. In this first approach a whole vector (HRL) was used for transformation with the idea to supply many possible targets of different length in one go to increase the probability for HR events for a proof of concept. Three different target sequences due to the given homologies were expected for HRL (Fig. 5B, detailed depiction in suppl. Fig. S2) and HRs could thus be present either as single or multiple events in one clone. Because diatoms are diplonts, in total four ligIV sites (on chromosome 7 and 6 ACS Paragon Plus Environment

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28), two fcpA sites, and at least two (NAS3) or seven (NAS4) antisense construct

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integrations (the AS construct itself or the vector backbone) could serve as targets. Analysis of the corresponding clones by southern blots gave hints that all three possible HR sequences served as locations for the integration of DNA (Fig. 6A-C, for complete blots see suppl. Fig. S4). In clone 4B derived from NAS4 this seemed to have occurred in one clone, and will thus be shown exemplarily first.

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HR in the ligIV locus should become visible by congruency of southern blot signals when probed with nat and 3’ligIV, probes detecting the nat resistance introduced by HRL and the ligIV locus, respectively. This was indeed the case in clone 4B (Fig. 6A and B). After NsiI restriction, a fragment of about 6.5 bp when probed with nat was also detected by 3’ligIV, i.e. when probing against the ligIV locus downstream of the target region. The smaller 3’ligIV positive signal between 4.5 and 6 kb after NsiI restriction was visible in clone 4B, but also in NAS4 and WT (Fig. S4), thus representing the WT locus (see suppl. Fig. S2). A HR using both flanking regions and replacing the target sequence by the nat cassette should have led to a fragment size of about 5.3 kb. Since the detected fragment of recombined ligIV is at about 6.5 kb, a larger insertion using the HRL vector must be suspected. Since the nat cassette has a size of 1.4 kb, the easiest explanation is a tandem insertion of this cassette. The upper most fragment detected by probing against nat at around 9.5 kb in clone 4B could also be detected by probing against ble (Fig. 6B), whilst no such fragment was present in NAS4. This indicates an integration of HRL into one of the at least seven antisense constructs present in NAS4. Further indication for antisense site(s) as HR target in clone 4B (as well as in 4A) is given in Fig. 6C: after SalI restriction and probing with ble the original knock-down mutant NAS4 displays a pattern of seven fragments ranging from around 2 kb to 9 kb, that is reduced to five fragments in clone 4A and one fragment in clone 4B. This can be explained by HR events which led to the replacement of ble genes that were present in the antisense constructs. The smallest nat positive fragment in clone 4B of about 3 kb (Fig. 6B) was not detected with any of our probes after using NsiI for DNA restriction. NsiI cuts once in the construct HRL, inside the 5’F region (see Fig. S2). In case of multiple insertions of HRL into ligIV, several nat positive fragments might occur that are not congruent with ligIV positive fragments. To verify this, MscI, an enzyme for which no restrictions sites are present in HRL, was used for DNA digestion (Fig. S5). The blot was probed against the (AS) antisense fragment, present in the AS construct and ligIV. The unchanged ligIV fragment at around 9.5 kb could be detected in WT, NAS4 and 4B. In NAS4, additional fragments from around 3 kb to 4.5 kb are present that are missing in 4B, supporting the idea of the use of antisense sites for HR. Clone 4B solely shows a double band at around 23 kb that could also be detected by re7 ACS Paragon Plus Environment


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probing against nat. This further supports the idea of a larger insertion of HRL elements into ligIV and/or an antisense construct and confirmed that all nat positive fragments of clone 4B can be explained by HR. When using SalI for DNA restriction and probing the blot against the 5’ region of fcpA (probe fcpA, Fig. 6C, compare suppl. Fig. S2) an additional fragment at around 6 kb below the original one at around 8 kb present in WT became visible in clone 4B that is indicative of a HR event on target fcpA. However, a replacement of the target sequence by nat between the two homologous elements pfcpA and tfcpA should

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have led to a fragment that was about 200 bp larger than the WT fragment, but instead a much smaller fragment was found. This fragment was not detectable when probing against nat (data not shown) and must therefore originate from another HR type. One explanation would be the elimination of nat together with further deletions during the HR mediated integration of HRL. The other HRL integrations at target ligIV and at the antisense location are thus sufficient to provide antibiotic resistance for

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this clone.

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It has been reported that subclones different from the original transformants can be obtained after transformation8. We carefully re-streaked all our clones and detected that in case of 4B two different subclones were analysed. One clone featured the nat

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positive fragment at around 9.5 kb (4B2), attributed to an HR event into an antisense site before, whereas in the other a 3 kb (4B1) fragment was detected. After having collected hints about the genetic localisations of the inserted constructs via southern blot, we tried to prove the HR events by PCR methods, but the exact determination of recombination processes turned out to be very susceptible to false positive results, as for example described by18. Using one primer binding in the genome and one in the construct is error prone due to strand annealing of complementary (homologous) sequences during PCR. Therefore, both primers needed to bind outside the flanking regions in the genome of HR target genes. However, this is still error prone in case of repetitive sequences, which often occurred due to tandem integrations. In addition, a predetermined breaking point in the middle of a tandem repeat sequence inside the nat gene led to an extremely decreased stability of larger amplificates (several kb). Altogether, this inhibited successful cloning approaches subsequent to PCR methods in most cases. Therefore, in order to reveal the genomic location of larger inserts where vector was included, plasmid DNA was recovered (see suppl. Fig. S7) and sequenced. In case of clone 4B1 two plasmids could be recovered after NsiI restriction. Sequencing indeed revealed that in clone 4B1 an HR into an antisense construct (Fig. 7A) with elimination of the elements ble and tfcpA* (compare Fig. 5B) had occurred. Since no 8 ACS Paragon Plus Environment

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further genomic parts could be found, it must be part of a multiple integration into an antisense site that can be fragmented using NsiI which cuts once in HRL. This could be further confirmed by another sequenced plasmid of clone 4B1 after NsiI based recovery (Fig. 7B). This plasmid consists of HRL only and must be a second part of a multiple integration of HRL. Further evidence for eliminating HR reactions could be found after sequencing of a PCR product from clone 4B1 (Fig. 7C and S6A, compare Fig. 5B) in which an integration of nat with an additional elimination of tfcpA had

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taken place.

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When using HRL to transform NAS4, the clones obtained all showed a different fragment pattern on southern blots (see Fig. 6A). In contrast, NAS3 derived clones were astonishingly similar. No congruency of fragments detected after NsiI digestion when probing with nat (Fig. 6A) and ligIV or fcpA specific probes (probes 3’ligIV and fcpA, see suppl. Fig. S2) was visible for the NAS3 derived clones 3A-E, but surprisingly the two nat-positive signals were identical in all clones tested. In order to

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test for integration at an antisense construct, DNA digested with SalI was also probed against ble, the antibiotic resistance introduced when creating the antisense mutants. However, no differences in fragment pattern between NAS3 and derived HRL mutants (two fragments at around 6 kb and 8 kb) could be observed (Fig. 6C). Thus, either no HR in an antisense construct had happened at those sites or the antisense construct targeted by HR did not contain ble due to incomplete integration. Therefore, in order to reveal the genomic location of the identical nat fragments in clones 3A-E (Fig. 6A, NsiI, probe nat), plasmid DNA was recovered from clone 3C using NsiI (see suppl. Fig. S7B, clones 1 and 3) or MscI (data not shown) and subsequently sequenced. The sequencing data revealed that a tandem integration of two complete HRL copies (i.e. the whole vector constructs) at chromosome 18 (Fig. 7D) between bp 308200 and bp 308213 had taken place. Concerning the NsiI derived plasmids, the upper fragment detected by probe nat with a size of 6.4 kb was only due to the HRL construct, whereas the lower fragment with a size of 5.8 kb additionally carried a part of the integrational site at chromosome 18. This could be confirmed to be identical for clone 3D by plasmid recovery using NsiI. Additionally, a MscI derived plasmid revealed the other end of the tandem HRL integration on chromosome 18 by sequencing (bp 308213). Thus, the two nat-positive fragments detected in all clones 3A-E after digestion of the DNA with NsiI are due to the insertion of two complete HRL copies in tandem on chromosome 18. After reprobing the SalI derived blot from Fig. 6C against the identified region on chromosome 18 (suppl. Fig. S8), all clones 3A-E showed an additional fragment at around 23 kb compared to the original one at around 9 kb present in NAS3. Thus, the specifity of this one-allelic integration can at first be explained by an antisense 9 ACS Paragon Plus Environment


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construct at this location serving as HR site, which got completely exchanged. However, no changes in fragment patterns when probing against ble between NAS3 and clones 3A-E could be detected. Thus, an incomplete antisense construct in NAS3 at chromosome 18 without the element ble has to be assumed. Plasmid recovery from NAS3 using several restriction enzymes could not reveal an antisense construct at the location on chromosome 18. If present, not only the ble sequence, but also necessary elements for plasmid replication in E. coli had not been (functionally) integrated. Although we could not prove that an incomplete AS construct was present in NAS3 on chromosome 18, the fact that all clones showed this specific insertion, but no insertion at this locus happened when using a construct without vector backbone for HR (HRT, see below), further supports this hypothesis. One explanation for this specific insertion might be a better accessibility of this part of the genome that already led to the insertion of an antisense construct. In contrast to NAS4, all clones derived from NAS3 showed this specific insertion on chromosome 18, which might also be an explanation for the high transformation efficiency of NAS3 with construct HRL. In addition to the HR into a presumable antisense construct on chromosome 18 present in all clones (3A-E), a specific change in fragment pattern could be found in clones 3A, B, C and E when probing the SalI derived blots against the putative promoter of ligIV (pligIV), a sequence that belongs to the target region (see suppl. Fig. S2). Besides the unchanged WT alleles at around 4.5 kb, an additional fragment at around 8 kb could be detected after probing the SalI derived blot (Fig. 6C). The additional fragments in clones 3A, B, C and E at around 8 kb are in ratio 1:1 to the original fragment at around 4.5 kb, thus probably representing a recombination on 2 of 4 alleles of ligIV. But neither after digestion with SalI, nor with NsiI or other restriction enzymes, congruencies between ligIV and nat detected fragments could be found, again arguing for a HR on target ligIV with concomitant exclusion of nat as shown for clone 4B in case of fcpA. The recombination on target ligIV (Fig. 6C, probe pligIV) was expected to further decrease the expression of ligIV, which could be

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confirmed using duplex PCR for clone 3C (Fig. 7E). Since southern blot intensities pointed to two of four alleles of ligIV being altered, only half of the ligIV expression

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compared to NAS3 was expected and found to be even lower with about 35%.

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In summary, the ligIV locus was shown to directly serve as location for HR by the phenotype of reduced ligIV expression in clone 3C, a notion that was underpinned by southern blot analysis. The fcpA locus and antisense sites were targeted as well, demonstrated by altered fragments or fragment patterns on southern blots compared to wild type and knock-down lines NAS3 and NAS4, by sequencing in clone 4B1 and by the specific integration on chromosome 18 in clones 3A-E also demonstrated by

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sequencing. Overall, all present integrations can be explained by HR mechanisms indicating a sufficient inactivation of NHEJ in the ligIV knock-down lines.

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The most important consequence of a ligIV knock-down is the drastic up-regulation of HR. After HRL integration analysis, a clear correlation between the number and length of homologous regions and targeting efficiency was found, in which a total homology of about 700 bp (HRL to fcpA) was quite inefficient (one out of seven clones). A length of about 2000 bp (HRL to ligIV) had a much better rate with five out of seven clones, while the highest rate has to be assumed for about 3000 bp (HRL to integrated antisense constructs, for all seven clones tested).

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Integration of HRT in NAS3 and NAS4

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After having demonstrated that HR events are increased in NAS3 and NAS4 we used more specific, but thus smaller constructs for more targeted HR. Expression cassettes HRT1 and HRT2 (Fig. 8) were used for transformation that both targeted tgl1 with a total homology of about 1 kb. In both cases the 5’ flanking region of 500 bases consisted of the putative promotor region of tgl1. Behind this promoter the nat

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gene was cloned. The difference between HRT1 and HRT2 concerned the 3’ flanking region, which consisted of part of the tgl1 coding region in case of HRT2, whereas the putative tgl1 terminator was used in case of HRT1. Tgl1 is a triacylglycerol lipase and its expression had been reduced by an antisense approach before19. However, since Tgl1 is not the only enzyme working in triacylglyceride breakdown19,20, homozygous knock-outs are not expected to be lethal. As expected, the

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transformation rate was much lower than in case of HRL with 0 (wild type), 1.5 · 10-8

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(NAS3) and 0.8 · 10-8 clones · µg-1 DNA · 108 cells-1 (NAS4) for HRT2 and even lower

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for HRT1 with 0 (wild type and NAS3) and 0.5 · 10-8 clones · µg-1 DNA · 108 cells-1

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(NAS4). 1.2 · 10-7 clones · µg-1 DNA · 108 cells-1.

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Two of those clones, T3B1 (derived from NAS3, HRT2) and T4B1 (derived from NAS4, HRT2), were found to be positive for a heterozygous recombination on target tgl1. After amplification of target tgl1 using the primer pair 5T_fw and 3T_rv (see Fig. 8A, 9A), fragments at around 6.5 kb were present in T3B1 and T4B1 additionally to the 4.5 bp fragments seen in WT, NAS3 and NAS4. Those were extracted and purified. The subsequent nested PCR (Fig. 9B) revealed an integration of nat into tgl1, because the 3’ region of tgl1 (3T) and nat could both be amplified. A replacement of the target sequence by nat between the flanking regions was expected to reduce the size of the tgl1 amplificate by 500 bp compared to the original tgl1 product, but instead an increase of about 2 kb could be found. After further PCR

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analysis and subsequent sequencing, the multiple integration of the HRT2 derived elements nat and 3FT in complete and incomplete versions and different orientations 11 ACS Paragon Plus Environment


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was revealed in clone T4B1 (Fig. 9C). The size of the sequenced product of about 1.8 kb fits to the recombinant fragments detected in T3B1 and T4B1 with an increase in size of about 2 kb.

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Phenotypical analysis of tgl1 mutants

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A functional disruption of one allele of tgl1 after HRT2 integration in clones T3B1 and

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T4B1 was expected to lead to an accumulation of triacylglycerols19. Using Nile Red staining and fluorescence spectroscopy (Fig. 9D), a 9-fold increased Nile Red fluorescence could be detected in clones T3B1 and T4B1 compared to wild type. Already NAS3 showed a slightly increased Nile Red fluorescence compared to wild type. This can be explained by the different size and shape of NAS3 cells (longer cells, partly tricornute) compared to wild type that could lead to a slightly different lipid content. Nevertheless, a clear accumulation of lipids resulting in higher Nile Red fluorescence was found as a consequence of HR on one allele of tgl1 in T3B1 and T4B1. This could be further confirmed by fluorescence microscopy (Fig. 9E). Cells of clones T3B1 and T4B1 showed either some large lipid bodies (T3B1) or numerous smaller ones (T4B1), whereas this was not the case in wild type cells. An overview of cells from wild type, NAS3, NAS4, T3B1 and T4B1 is shown in suppl. Fig. S9. Nile Red fluorescence in living cells (shown by chlorophyll autofluorescence) caused by the increased formation of lipid bodies could only be detected in clones T3B1 and T4B1.

380

Outlook

381

Integration of foreign DNA by HR can occur in numerous types like replacement, inversion, deletion or dimerization21 dependent on the different integration types22 present in eukaryotes. The best known type of integration of foreign DNA by HR is the replacement of a target sequence between two flanking regions by e.g. an expression cassette containing an antibiotic resistance gene. Other types of integration can occur if for example only one flanking region is used or secondary structures between involved DNA molecules (loops) are formed during the recombination process.

361

366 367 368 369 370 371 372 373 374 375 376 377 378

382 383 384 385 386 387 388 389 390 391 392 393 394 395

HR events in P. tricornutum at predetermined target sites indeed turned out to be complex, i.e. besides the expected exchange of the target by the nat cassette using the flanking regions, larger integrations of DNA, complete exchange of the target by the constructs as well as deletions of elements could be found. There are different strategies available for gene targeting in P. tricornutum by nucleases, which can be used to mediate HR events8,9,11. In these cases our results can be helpful to identify possible complex HR reactions. Overall, tandem integrations seem to be a preferred 12 ACS Paragon Plus Environment

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396

type in P. tricornutum, which could make gene replacement strategies very efficient if

397

two recombinant gene copies can replace the original one. In case of using HRL, a construct including a total vector, a higher probability of HR in the vector DNA of the antisense site was observed due to the length of the homologous regions. This might imply a disadvantage, but could be used to also remove the ligIV antisense effect.

398 399 400 401 402 403 404 405 406 407 408 409 410

Another possibility to avoid starting from mutants that have nevertheless the antisense constructs inserted at random locations in the genome would be the episomal strategy introduced by10 to create ligIV mutants. By using episomes that are transferred by conjugation but remain stable in diatoms the danger of having negative effects due to the insertion of antisense constructs will be avoided. Compared to other genome based strategies for gene targeting, the major advantages of our presented strategy lie in an efficient inactivation of NHEJ and thus an increase of the probability for HR and, thereby, reduction of unspecific integrations. Furthermore, off-target restrictions can be completely excluded because

414

DNA nucleases were not used. The lack of unspecific integrations (and restrictions) causing physiological problems was reflected in high transformation rates. Both basic research and biotechnology could profit from the creation of such mutants, which could be further optimised by simultaneously targeting genes and replacing the inserted antisense constructs, i.e. restoring the ligIV expression back to normal.

415

Conclusion

416

In the present study, we can show that unspecific integrations are prevalent when foreign DNA is incorporated into the genome when NHEJ is working, independent of the constructs used. Knock-down of the LigIV homolog, which was characterized as a DNA ligase of type IV, inhibited the NHEJ mechanism and created the possibility to integrate DNA via HR at specific locations.

411 412 413

417 418 419 420 421 422 423 424 425 426 427 428 429 430 431

In a first approach HRL was used, a construct with three possible target sites: the ligIV locus itself, the vector backbone of the antisense construct and fcpA. All of them served as targets, whereby the probability was determined by the number of sites and the length of homology. The small number and short length of the homologies restricted the HR efficiency when using a construct without vector backbone to specifically target one gene, i.e. HRT targeting tgl1. Different types of events by HR were observed: i) insertions of elements from the constructs in between homologous regions, mostly as tandems (e.g. nat into ligIV in clone 4B), ii) only partial integrations (e.g. HR into the ligIV locus in clones 3A, B, C and E without the nat gene) or even major deletions due to the HR (e.g. HR into the fcpA locus in clone 4B) and iii) complete substitution of targets (e.g. HR into 13 ACS Paragon Plus Environment


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chromosome 18 in clones 3A-E). Thereby, the kind of HR seemed to be independent of the target sequence used. The effect of specific gene targeting and consequent disruption of the functionality could be demonstrated for clone 3C, in which the expression of ligIV was decreased, and for T3B1 and T4B1 that also revealed the expected phenotype of lipid accumulation.

437

Methods

438

Cell Culture

439

444

P. tricornutum (strain UTEX 646) was cultivated in 25 or 50 ml batch cultures in artificial seawater medium (ASP23) at 16 - 18 °C while exposed to a cycle of 16 h of white light (40 µmol photons m-2s-1) and 8 h of darkness. Determination of cell number for transformations was done using a Thoma chamber. Selection of clones was carried out on 1.5 % (w/v) Agar-ASP plates containing 75 µg/ml Zeocin™ (InvivoGen) or 100 µg/ml Nourseothricin (Werner Bioagents).

445

For plasmid DNA replication, E. coli strain XL1-Blue (Stratagene) was transformed by

446

449

heat shock with the respective plasmid DNA and selection was done for 16 hours at 37 °C on 1.3 % (w/v) Agar-LB (1 % tryptone, 0,5 % yeast extract and 0,5 % NaCl) plates containing 100 µg/ml Ampicillin (AppliChem). Afterwards, clones were cultivated in LB medium with 100 µg/ml Ampicillin for 16 hours at 37 °C.

450

LigIV homolog in P. tricornutum

451

Structural model

452

Two identical copies of a putative LigIV homolog (NCBI Database: www.ncbi.nlm.nih.gov; gene ID: EEC48802.1 and EEC43238.1, on chromosome 7: Phatr2|chr_7:237352-241342; on chromosome 28: Phatr2|chr_28:18412-22402) were found in the predicted NHEJ pathway (KEGG database: http://www.genome.jp/kegg-bin/show_pathway?pti03450) of P. tricornutum. The

433 434 435

440 441 442 443

447 448

453 454 455 456 457 458 459

predicted sequence contains a DNA and ATP binding motif typical for DNA ligases. The amino acid sequence of LigIV from P. tricornutum was modelled against the

460 461

Purification of recombinant LigIV_his and DNA ligation assay

462

For heterologous expression of LigIV with a tenfold N-terminal His-tag (LigIV_his), ligIV was amplified by Phusion polymerase (Thermo Scientific) in a fusion PCR reaction24 using primers L4_fw, L4_rv, fw_fus and rev_fus (primer sequence see suppl. Table S1), to exclude the intron. The amplification product was cloned into

463 464 465

human

LigIV


by

phyre217

known structure of the (www.sbg.bio.ic.ac.uk/~phyre2).

software

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466

vector pJET1.2 and sequenced. After reamplification, ligIV was cloned into vector

467

pET302/NT-His (Invitrogen) by a hot fusion reaction25 using the primers L4Nhis_fw and L4Nhis_rv (primer sequence see suppl. Table S1), which included additional nucleotides to enlarge the His6 to a His10 tag. The constructs were used to transform E. coli XL1 blue. After sequencing, E. coli BL21 Codon+ cells (Stratagene) were

468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495

transformed by heat shock with this construct or the respective empty vector (EV) control (pET302/NT-His). For protein expression, cells were grown in 2 l DYT medium26 and supplemented with 100 µg/mL ampicillin and 34 µg/mL chloramphenicol (Sigma). Expression was induced at an OD600 nm of 0.8 with 1 mM isopropyl β-D-1-thiogalactopyranoside and carried out at 18 °C for 5 h. Cells were harvested at 3019 x g and 4 °C for 10 min and cell disruption was done in lysis buffer (50 mM sodium phosphate pH 8; 300 mM NaCl) using a French Press (5 cycles at 1000 p.s.i.; Thermo Electron Corporation). The lysates were centrifuged at 39121 x g and 4 °C for 20 min and the supernatants were loaded on Ni-NTA columns (Chelating SepharoseTM FastFlow, GE Healthcare). Elution was done with 300 mM imidazole in 50 mM sodium phosphate pH 8 and 1 M NaCl, and afterwards the eluates of LigIV_his and the respective EV control were concentrated by dialysis as done by27 to 1 ml. Detection of recombinant LigIV_his was done by SDS-Page28 and immunoblotting using 500 µl LigIV_his, 700 µl EV eluate and anti-his antibody (1:3000, Sigma-Aldrich). In order to investigate a possible DNA ligation capability of LigIV_his, its ability to religate linearized vector-DNA was investigated. For this purpose, vector pPha-T1 (accession number AF 219942) was linearized with XbaI (cuts in the multiple cloning site) at 37°C (Thermo Scientific) for several hours and 30 ng were used for each ligation approach. 50 % (v/v) of total LigIV_his eluate and 70 % (v/v) of total EV eluate were used in ligation assays (20 µl). T4 DNA ligase (5 U, Thermo Scientific) served as positive control and H2O as negative control. The reaction was carried out in 40 mM Tris pH 8, 5 mM MgCl2 and 0.5 mM ATP. Incubation was done at 18 °C up to one hour, followed by heat inactivation and transformation of E. coli XL1 blue.

496

Clones were selected by the acquired antibiotic resistance and counted in comparison to the water and empty vector controls to reveal the rate of DNA ligation.

497

Isolation of nucleic acids

498

Isolation of genomic DNA from P. tricornutum

499

Genomic DNA from P. tricornutum UTEX 646 was isolated using a modified protocol

500

after29,30. Cell disruption was done using liquid nitrogen, mortar and pestle till a fine powder remained. After adding a 2x cetrimonium bromide (CTAB30) solution, DNA was extracted with isoamyl alcohol/chloroform (24:1, Roth) followed by an additional phenol/isoamyl alcohol/chloroform (25:24:1, Roth) and chloroform extraction.

501 502 503



504

509

Afterwards, precipitation was achieved using isopropanol / 0.3 M sodium acetate pH 5 at -20 °C for one hour. The precipitate was two times washed with 70 % ethanol (Roth) and after drying resuspended in 10 mM Tris pH 8 (VWR). Remaining RNA was degraded with 5 µl RNAse A (10 mg/ml, Thermo Scientific) for 1 h at 37 °C followed by a phenol/isoamyl alcohol/chloroform (25:24:1) and chloroform extraction. Precipitation and resuspending was done as described above.

510

Isolation of RNA from P. tricornutum

511

In case of isolation of total RNA, a modified protocol after31 was used. Cell disruption

512

was done with a Tissue Lyser LT (Qiagen) at 50 Hz for 15 minutes in Roti® -Aqua-

513

P/C/I (Roth), and afterwards 1 volume of 0.1 % DEPC-H2O (autoclaved, Roth) was

514

added. After extraction, an additional Roti® -Aqua-P/C/I and chloroform extraction

515

518

was carried out followed by a 2.5 M LiCl2 (VWR) precipitation at -20 °C over night. The precipitate was two times washed with 70 % ethanol in 0.1 % DEPC-H2O, dried and afterwards resuspended in 0.1 % DEPC-H2O. Remaining DNA was degraded using 3 µl DNAseI (Thermo Scientific) for 3 h at 37 °C. RNA was purified using

519

Roti® -Aqua-P/C/I and chloroform, followed by a precipitation using 2.5 M LiCl2 at -20

520 522

°C over night and washed as described above. 1 µg of total RNA was transcribed into cDNA using RevertAid Transcriptase (Thermo Scientific) and Oligod(T)18 primer (biomers).

523

Plasmid Isolation from E. coli

524

529

Plasmid isolation was done using the GeneJET Plasmid Miniprep Kit (Thermo Scientific) or using DNA binding columns (Roth). The latter were used in the same way as outlined in the supplier’s instructions for the kit. Resuspension was done in 130 µl 50 mM glucose, 25 mM Tris pH 8, 10 mM EDTA and 0.25 % (w/v) RNAse A (Sigma). Cell lysis was achieved using 350 µl of 0.2 M NaOH and 1 % SDS (w/v) and neutralization using 230 µl 2.5 M potassium acetate pH 4.8.

530

Concentration of nucleic acids

531

534

Nucleic acid concentration was determined at 260nm using a Nanodrop ND-1000 (Thermo Scientific) or by measuring the pixel content with ImageJ (https://imagej.nih.gov/ij/) on agarose gels in comparison to marker GeneRuler DNA Ladder Mix (Thermo Scientific).

535

Amplifications, restriction, cloning and sequencing

536

For PCR test amplifications (simple, duplex and nested PCR) the taq polymerase protein was overexpressed in E. coli DH5α and enriched according to32. After

505 506 507 508

516 517

521

525 526 527 528

532 533

537


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538


546

successful testing, 1 µl of taq eluate was used in PCR-Reactions with a total volume of 15 µl. As a reaction buffer, 10 mM Tris pH 8.3, 40 mM KCl and 4 mM MgCl2 was chosen and 10 % (v/v) loading dye (xylenol orange in 50 % (v/v) glycerol) as well as 5 - 10 pmol of each primer, 1.6 µl of 5 mM dNTPs (each) and 0.26 M Betain were added. After an initial denaturation at 95 °C for five minutes, 20 - 35 cycles were carried out with a denaturation step at 95 °C for 30 s, an annealing step at about -2 °C to -5 °C of the calculated melting temperature (by supplier biomers) for 30 s and an elongation step at 72 °C for 60 s/kb of the expected product size. A final elongation step was applied at 72 °C for ten minutes.

547

For cloning purposes, tgl1 locus analysis and the creation of probe DNA for southern

548

blots, the proof reading Phusion polymerase (Thermo Scientific) was used according to the supplier’s instructions in a 3-step protocol. Annealing temperatures were chosen as described for taq amplifications and elongation steps were done at 72 °C for 30 s/kb of the expected product size.

539 540 541 542 543 544 545

549 550 551 552 553 554 555 556 557 558 559 560 561 562

Purification of PCR products was done using the GeneJET Gel Extraction Kit (Thermo Scientific) or DNA binding columns (Roth). Purification using the latter was achieved by melting extracted DNA-containing agarose pieces at 60 °C in 5 - 10 volumes 6 M NaI for 10 minutes. After subsequent loading, columns were washed three times with washing buffer (70 % ethanol in 20 mM Tris pH 7.4; 1 mM EDTA; 50 mM NaCl, ice cold) and dried by centrifugation. Elution was done using 60 °C prewarmed 10 mM Tris pH 8. For sequencing and further cloning purposes, vector pJET1.2 (CloneJET PCR cloning Kit, Thermo Scientific) was used and all sequencing reactions were done by GATC Biotech (Konstanz, Germany).

563

All restriction enzymes were purchased from Thermo Scientific and used according to the supplier’s instructions.

564

Southern Blot

565

Digoxygenin (DIG) labelled probe DNA for southern blots33 was either created using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche), or DIG labelled UTP (Jena Bioscience), klenow fragment (Thermoscientific) and random hexamer primer (Thermo Scientific). Probe DNA was amplified by Phusion polymerase using specific primers (see suppl. Table S1). Afterwards, probe DNA was purified by gel extraction. 1 µg of purified probe DNA was labelled at 37 °C over night and again purified. Concentration of labelled probe DNA was estimated on dot blots in comparison to labelled control DNA (included in the kit). Labelled probe DNA was used at a concentration of 5 ng/ml. 4 - 6 µg genomic DNA per cell line were digested

566 567 568 569 570 571 572 573



574

581

with NsiI (or isochizomere Mph1103I), SalI or MscI for 16 hours at 37 °C, followed by heat inactivation. Both NsiI and Mph1103I recognize and cut the same target sequence and are therefore both referred as NsiI in the following. Remaining RNA was degraded with 0.5 µl RNAse A (10 mg/ml) for 30 minutes at 37 °C and DNA separated on 1 % agarose gels. To estimate fragment size, DNA Molecular Weight Marker II (DIG-labelled, Sigma) was used. Afterwards, DNA was transferred on amphoteric nylon membranes (Porablot NY amp, Macherey-Nagel) using vacuum blotting and standard solutions34.

582

Table 1: Probes and corresponding primers used for southern blots (for primer sequence see suppl. Table S1).

575 576 577 578 579 580

probe 18 (521 bp) nat (593 bp) ble (395 bp) pligIV (267 bp) AS (242 bp) S2 (943 bp) 3’ligIV (217 bp) fcpA (1001 bp)

forward primer 18_fw3

reverse Primer 18_rv

nat_fw

nat_rv

ble_fw

ble_rv

SB_fw

SB_rv

sAS_fw

sAS_rv

S2_fw

S2_rv

3DL_fw

3DL_rv

lhcf1_fw

lhcf1_rv

target chromosome 18 bp 307680 - 308200 nat gene (nourseothricin resistance) ble gene (zeocin resistance) putative ligIV promoter (pligIV) from bp -330 to -64, see Fig. S2 antisense fragment of ligIV from bp 68 - 290, see Fig. S2 ligIV cds from bp 2015 - 2957, see Fig. S2 ligIV cds and 3’UTR from bp 3957 to 4173, see Fig. S2 5’ region up-stream of the fcpA promoter from bp -1439 to -440, see Fig. S2

583 584

A true to scale schematic representation of the respective loci of ligIV and fcpA, as

585

588

well as overviews of respective constructs with used restriction sites and target sequences of probes is given in suppl. Fig. S2. Pixel content of detected fragments on southern blots was measured using the software imageJ (https://imagej.nih.gov/ij/).

589

Generation of ligIV knock-down mutants

590

595

The antisense DNA (223 bp) was amplified from ligIV (Fig. 1) of P. tricornutum using the primers sAS_fw and sAS_rv (primer sequence see suppl. Table S1), thereby creating a SalI and Xbal restriction site. The fragment was cloned into the vector pJET1.2 and sequenced. Using the newly created restriction sites for XbaI and SalI, the antisense fragment was cloned inversely into the target vector pPha-T1 (accession number AF 219942). P. tricornutum cells were transformed according to6

596

using 1 µg vector DNA per shot on 108 cells.

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597

Characterization of ligIV knock-down mutants

598

Mutants were screened by measuring the relative mRNA content of ligIV after

599

reverse transcription in duplex PCR reactions using the primers UDL4_fw and UL4_rv for ligIV (both binding in the 3’ UTR) and H43_fw and H43_rv (for primer sequence see suppl. Table S1) for comparison with the housekeeping gene h4. Three independent RNA isolations per each cell line were used and each replicated three times. Pixel content of fragments on agarose gels was measured using the software imageJ (https://imagej.nih.gov/ij/). Two mutants, NAS3 and NAS4, were selected for further experiments. The same duplex PCR method was also used to estimate ligIV mRNA levels in mutants created by HR.

600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615

UV sensitivity was examined by illuminating plated cells of wild type and ligIV knockdown mutants NAS3 and NAS4 with 1.5 Joule/cm2 for 5 minutes in a UV crosslinker (Herolab). Plates that were not exposed to UV radiation served as controls. Afterwards, all plates were incubated for six weeks and colonies were counted. Survival rate was calculated as the number of grown colonies of UV-treated cells in relation to the colonies on control plates. As one physiological parameter for determining the photosynthetic fitness, the fluorescence parameter FV/Fmax was measured in a FL 3000 Fluorometer (PSI, Czech Republic) with three measurements per cell line. Cultures of wild type and ligIV

619

knock-down mutants NAS3 and NAS4 were diluted in 2 ml ASP media to 5 x 106 cells/ml, 4 mM KHCO3 was added and cells were adapted to darkness for 20 minutes. F0 was measured without actinic illumination and afterwards Fmax was determined using saturating light flashes. FV was calculated as Fmax - F0.

620

Construction and transformation of HR construct HRL

621

Construct HRL for HR on targets ligIV (total homology 2002 bp), fcpA (total homology 705 bp) and previously integrated antisense constructs (maximum total homology 3116 bp, dependent on completeness of antisense construct integration) was designed in the following procedure: the nourseothricin resistance gene (nat) was

616 617 618

622 623 624 625 626 627 628 629 630 631 632

amplified from the vector pPha-T1_nat35 with the primers nat_fw and nat_rv (primer sequence see suppl. Table S1), cloned into vector pJET1.2 and sequenced. Afterwards the nat gene was cut out with NheI and XhoI and cloned into the multiple cloning site of vector pPha-T1. The vector now contained the nat gene under control of the promoter and terminator of fcpA. This cassette (kon_nat) was amplified with primers kon_fw and kon_rv (primer sequence see suppl. Table S1). The 5’ and 3’ flanking regions 5’F and 3’F targeting ligIV (Fig. 1, S2) were amplified from genomic DNA of P. tricornutum. The 5’ 19 ACS Paragon Plus Environment


633 634 635 636 637

flanking region was amplified with primers 5F_fw and 5F_rv, the 3’ flanking region was amplified with 3F_fw and 3F_rv (primer sequence see suppl. Table S1). All products were separately cloned into vector pJET1.2 and sequenced. Finally, all products were cloned together into pJET1.2, leading to the final construct HRL.

642

P. tricornutum wild type (WT) and ligIV knock-down mutants NAS3 and NAS4 were transformed according to6 with 1 µg DNA per shot on 108 cells. Prior to and after transformation cells were kept in darkness for 24 hours. Clones were picked, resuspended in 50 - 200 µl ASP medium and one part was replated on antibiotic containing plates to receive subclones, while the other part was used to inoculate liquid cultures.

643

Plasmid DNA recovery from P. tricornutum

644

Integrated plasmid DNA was recovered from P. tricornutum after21. This method can only be used to reveal its genomic location, if elements for plasmid DNA replication in E. coli like origin of replication and antibiotic resistance are present. 1 µg of isolated genomic DNA from P. tricornutum clone 3C and 3D was digested at 37 °C over night

638 639 640 641

645 646 647 648

652

with NsiI (for 3C, 3D and 4B1 that introduces cuts in the genome and once in the inserted HRL construct, see suppl. Fig. S2) or with MscI (for 3C, doesn’t cut in the inserted HRL construct), and inactivated at 80 °C for 20 minutes. Religation was done at 16 °C over night with T4 DNA Ligase (Thermo Scientific) and afterwards E. coli was transformed.

653

Construction and transformation of HR construct HRT

654

Constructs for a HR on a single target, tgl119 (NCBI gene ID: XP_002184517.1,

655

Phatr2|chr_24:282391-284055), were made according to the following procedure: The gene for nourseothricin resistance nat was cloned into vector pJET1.2 and sequenced (see construction of HRL). As a 5’ flanking region the putative promoter of tgl1 (ptgl1) was amplified with primers ptgl_fw and ptgl_rv (primer sequence see suppl. Table S1) from genomic DNA of P. tricornutum, cloned into vector pJET1.2 for sequencing, cut out with XbaI and NheI and cloned in front of nat into vector pJET1.2.

649 650 651

656 657 658 659 660 661 662

667

For the first HR construct HRT1, the putative terminator of tgl1 (ttgl1) was chosen as the 3’ flanking region. Therefore, ttgl1 was amplified from genomic DNA of P. tricornutum with primers ttgl_fw and ttgl_rv (primer sequence see suppl. Table S1), cloned into vector pJET1.2, sequenced, cut out with SalI and XhoI and cloned behind nat. In case of the second construct HRT2, the 3’ flanking region was chosen to be within tgl1 (3FT). 3FT was amplified with primers 3FT_fw and 3FT_rv (primer

668

sequence see suppl. Table S1), cloned, sequenced, cut out with SalI and XhoI and

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669 670 671 672 673


cloned behind nat as for ttgl1. Both expression cassettes HRT1 and HRT2 contained a total homology of about 1 kb to tgl1. For transformation of P. tricornutum, plasmid DNA of both constructs was isolated from E. coli. The expression cassettes HRT1 and HRT2 were purified after restriction

678

(HRT1: NotI, XbaI; HRT2: XhoI, XbaI) by gel extraction. Concentration was determined by ImageJ on an agarose gel by comparison with the marker GeneRuler DNA Ladder Mix (Thermo Scientific). P. tricornutum wild type and ligIV knock-down mutants NAS3 and NAS4 were transformed with 130 ng purified expression cassettes HRT1 and HRT2 per shot on 108 cells as described for the transformation of HRL. The use of expression cassettes should enable HR on target tgl1 without

679

other HR events due to only one homology present.

680

PCR Analysis of tgl1 mutants

681

50 ng of genomic DNA from wild type, NAS3, NAS4, T3B1 and T4B1 were used as

682

template for PCR with Phusion polymerase (primers 5T_fw, 3T_rv, primer sequence see suppl. Table S1). Fragments at 6.5 kb (from T3B1, T4B1) were purified with an elution volume of 10 µl and 1 µl of the eluate was used for nested PCR to test for an integration of nat (from HRT2) into tgl1. Therefore, the primers nat_fw and nat_rv for amplification of nat as well as 3T_fw and 3T_rv for amplification of the 3’ region behind tgl1 (primer sequence see suppl. Table S1) were used. Additionally, primer

674 675 676 677

683 684 685 686 687 688 689

nat_fw was used to amplify the HRT2 derived cassette of T4B1 (50 ng) by Phusion Polymerase.

690

Phenotypical analysis of tgl1 mutants

691

Clones T3B1 and T4B1 were tested positive for a HR on one allele of tgl1 and thus an accumulation of triacylglycerols19 in form of lipid bodies was expected. To stain lipids in vivo, the fluorescent dye Nile Red was used36. Therefore, pre-cultures of wild type, NAS3, NAS4, T3B1 and T4B1 were grown for two weeks without the use of antibiotics. Afterwards, one culture per cell line was inoculated to an OD750nm = 0.125 and cultivated for four days. Cultures were taken at the end of the dark-phase and all following steps were done with minimal light exposure.

692 693 694 695 696 697 698 699 700 701 702 703

Nile Red fluorescence after binding to lipids was measured as described by23 in a Jasco FP-6500 fluorescence spectrophotometer. Three technical replicates per cell line were diluted to an OD750nm = 0.3 and incubated with 4 µl Nile Red solution (250 mg/l in acetone, Sigma) for 15 minutes. Excitation was done at 490 nm and fluorescence measured at 585 nm. ASP-Medium containing the same amount of Nile Red was used as a blank measurement. 21 ACS Paragon Plus Environment


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For fluorescence microscopy, 1 ml of culture per cell line was used and after natural sedimentation, 900 µl of medium was removed. Afterwards, 4 µl Nile Red solution (25 mg/ml in acetone) was added and cells were incubated for about 20 minutes. Images were taken using a Zeiss LSM 510 META confocal fluorescence microscope. Excitation was achieved at 488 nm, Nile Red fluorescence was detected using a BP 505 - 530 filter and chlorophyll autofluorescence using a LP 530 filter. Besides overview images for all cell lines, z-stack images of selected cells of wild type, T3B1 and T4B1 were recorded using a 63x magnification objective as well as digital magnification and used to reconstruct 3D models.

713

Supporting Information

714 715

The supporting information is available free of charge on the ACS website at http://pubs.acs.org

716

Table S1; Figures S2-10

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Author information

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Corresponding author

721

*E-mail: [email protected]

722

Author contributions

723

C.B. initiated the project and supervised its progress. M.A. planned and performed all major experiments as part of his PhD thesis and supervised the recombinant LigIV studies by J.K. and physiological analysis of ligIV knockdown mutants by O.A.. The manuscript was written by M.A., C.B. and J.K..

724 725 726 727 728 729 730 731 732

Conflict of interest statement: The authors declare no competing financial interests. Acknowledgements. We would especially like to thank Fabian Fischer (ETH Zürich), Andrea Hamann, Kehel Wohra, Uwe Bodensohn, Markus Fauth, Ulrike Eilers and Matthias Schmidt (all from Goethe University Frankfurt) for their help with the experiments.

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References 22 ACS Paragon Plus Environment

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1.

Kroth, P. (2007) Molecular biology and the biotechnological potential of diatoms. Adv. Exp. Med. Biol. 616, 23–33.

737 738

2.

Bozarth, A., Maier, U. G., Zauner, S. (2009) Diatoms in biotechnology: modern tools and applications. Appl. Microbiol. Biotechnol. 82(2), 195–201.

739 740 741 742

3.

Bowler, C., Allen, A.E., Badger, J.H., Grimwood, J., Jabbari, K., Kuo, A., Maheswari, U., Martens, C., Maumus, F., Otillar, R.P. et al. (2008) The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456(7219), 239–244.

743 744 745

4.

Maheswari, U., Montsant, A., Goll, J., Krishnasamy, S., Rajyashri, K.R., Patell, V.M., Bowler, C. (2005) The diatom EST database. Nucleic Acids Res. 33, D344–D347.

746 747 748

5.

Maheswari, U., Mock, T., Armbrust, E.V., Bowler, C. (2009) Update of the diatom EST database: a new tool for digital transcriptomics. Nucleic Acids Res. 37, D1001–D1005.

749 750 751

6.

Apt, K.E., Kroth-Pancic, P.G., Grossman, A.R. (1996) Stable nuclear transformation of the diatom Phaeodactylum tricornutum. Mol. Gen. Genet. 252(5), 572–579.

752 753 754

7.

Riso, V. de, Raniello, R., Maumus, F., Rogato, A., Bowler, C., Falciatore, A. (2009) Gene silencing in the marine diatom Phaeodactylum tricornutum. Nucleic Acids Res. 37(14), e96.

755 756 757 758

8.

Daboussi, F., Leduc, S., Maréchal, A., Dubois, G., Guyot, V., Perez-Michaut, C., Amato, A., Falciatore, A., Juillerat, A., Beurdeley, M., et al. (2014) Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology. Nat. Commun. 5, 3831.

759 760 761

9.

Nymark, M., Sharma, A.K., Sparstad, T., Bones, A.M., Winge, P. (2016) A CRISPR/Cas9 system adapted for gene editing in marine algae. Sci. Rep. 6, 24951.

762 763 764 765 766

10.

Slattery, S.S., Diamond, A., Wang, H., Therrien, J.A., Lant, J.T., Jazey, T., Lee, K., Klassen, Z., Desgagné-Penix, I., Karas, B.J., Edgell D.R. (2018) An expanded plasmid-based genetic toolbox enables Cas9 genome editing and stable maintenance of synthetic pathways in Phaeodactylum tricornutum. ACS Synth. Biol. 7(2), 328–338.

767 768 769 770 771

11.

Weyman, P.D., Beeri, K., Lefebvre, S.C., Rivera, J., McCarthy, J.K., Heuberger, A.L., Peers, G., Allen, A.E., Dupont, C.L. (2015) Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis. Plant Biotechnol. J. 13(4), 460–470.

772 773 774

12.

Fu, Y., Foden, J.A., Khayter, C., Maeder, M.L., Reyon, D., Joung, J.K., Sander, J.D. (2013) High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31(9), 822–826.



775 776 777

13.

Stukenberg, D., Zauner, S., Dell’Aquila, G., Maier, U.W. (2018) Optimizing CRISPR/Cas9 for the diatom Phaeodactylum tricornutum. Front. Plant Sci. 9, 740.

778 779 780 781

14.

Ishibashi, K., Suzuki, K., Ando, Y., Takakura, C., Inoue, H. (2006) Nonhomologous chromosomal integration of foreign DNA is completely dependent on MUS-53 (human Lig4 homolog) in Neurospora. Proc. Natl. Acad. Sci. USA 103(40),14871–14876.

782 783 784

15.

Schorsch, C., Köhler, T., Boles, E. (2009) Knockout of the DNA ligase IV homolog gene in the sphingoid base producing yeast Pichia ciferrii significantly increases gene targeting efficiency. Curr. Genet. 55(4), 381–389.

785 786 787 788

16.

Myung, K., Ghosh, G., Fattah, F.J., Li, G., Kim, H., Dutia, A., Pak, E., Smith, S., Hendrickson, E.A. (2004) Regulation of telomere length and suppression of genomic instability in human somatic cells by Ku86. Mol. Cell. Biol. 24(11), 5050–5059.

789 790 791

17.

Kelley, L.A., Mezulis, S., Yates, C.M., Wass, M.N., Sternberg, M.J.E. (2015) The Phyre2 web portal for protein modelling, prediction and analysis. Nat. Protoc. 10(6), 845–858.

792 793

18.

Won, M., Dawid, I.B. (2017) PCR artifact in testing for homologous recombination in genomic editing in zebrafish. PLoS ONE 12(3), e0172802.

794 795 796

19.

Barka, F., Angstenberger, M., Ahrendt, T., Lorenzen, W., Bode, H.B., Büchel, C. (2016) Identification of a triacylglycerol lipase in the diatom Phaeodactylum tricornutum. Biochim. Biophys. Acta 1861(3), 239–248.

797 798 799 800

20.

Trentacoste, E.M., Shrestha, R.P., Smith, S.R., Glé, C., Hartmann, A.C., Hildebrand, M., Gerwick, W.H. (2013) Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth. Proc. Natl. Acad. Sci. USA 110(49), 19748–19753.

801 802 803 804

21.

Steiner, S., Wendland, J., Wright, M.C., Philippsen, P. (1995) Homologous recombination as the main mechanism for DNA integration and cause of rearrangements in the filamentous ascomycete Ashbya Gossypii. Genetics 140(3), 973–987.

805 806 807

22.

Sung, P., Klein, H. (2006) Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat. Rev. Mol. Cell. Biol. 7(10), 739–750.

808 809

23.

Provasoli, L., McLaughlin, J.J., Droop, M.R. (1957) The development of artificial media for marine algae. Arch. Mikrobiol. 25(4), 392–428.

810 811

24.

Yon, J., Fried, M. (1989) Precise gene fusion by PCR. Nucleic Acids Res. 17(12), 4895.

812 813 814

25.

Fu, C., Donovan, W.P., Shikapwashya-Hasser, O., Ye, X., Cole, R.H. (2014) Hot Fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase. PLoS ONE 9(12), e115318.


Page 24 of 33

Page 25 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


815 816

26.

Miller, J.H. (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, NY.

817 818 819

27.

Nick McElhinny, S.A., Snowden, C.M., McCarville, J., Ramsden, D.A. (2000) Ku recruits the XRCC4-ligase IV complex to DNA ends. Mol. Cell. Biol. 20(9), 2996–3003.

820 821

28.

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259), 680–685.

822 823 824

29.

Borges, A., Rosa, M.S., Recchia, G.H., Queiroz-Silva, J.R. de, Bressan, E. de A., Veasey, E.A. (2009) CTAB methods for DNA extraction of sweetpotato for microsatellite analysis. Sci. Agric. 66(4), 529–534.

825 826 827 828

30.

Kira, N., Ohnishi, K., Miyagawa-Yamaguchi, A., Kadono, T., Adachi, M. (2015) Nuclear transformation of the diatom Phaeodactylum tricornutum using PCRamplified DNA fragments by microparticle bombardment. Mar. Genomics 25, 49–56.

829 830 831

31.

Chomczynski, P., Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162(1), 156–159.

832 833 834

32.

Desai, U.J., Pfaffle, P.K., (1995) Single-step purification of a thermostable DNA polymerase expressed in Escherichia coli. Biotechniques 19(5), 780– 782, 784.

835 836

33.

Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98(3), 503–517.

837 838

34.

Sambrook, J., Fritsch, E.F., Maniatis, T. (1989) Molecular cloning: A laboratory manual, 2nd Edition. Cold Spring Harbor Laboratory Press, New York.

839 840 841

35.

Eilers, U., Bikoulis, A., Breitenbach, J., Büchel, C., Sandmann, G. (2016) Limitations in the biosynthesis of fucoxanthin as targets for genetic engineering in Phaeodactylum tricornutum. J. Appl. Phycol. 28(1), 123–129.

842 843

36.

Greenspan, P. (1985) Nile red: a selective fluorescent stain for intracellular lipid droplets. J. Cell. Biol. 100(3), 965–973.

844



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Figure 1. True to scale schematic representation of the two identical ligIV gene copies present in P. tricornutum on chromosome 7 and 28. The antisense fragment against ligIV (AS, white arrow) is depicted as well as the flanking regions (5’F, 3’F, grey boxes) used for HR targeting of the ligIV coding region (dotted), and the putative promoter (pligIV) of ligIV.

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Figure 2. The LigIV coding sequence of P. tricornutum was modelled on the known structure of human DNA ligase IV by phyre2. Alignment confidence is shown in (A), quality assessment in (B), presence of the predicted DNA binding site is shown in red in (C), and the predicted ATP binding site in red in (D). Recombinant LigIV_his and the corresponding empty vector control (EV) were purified by Ni-NTA after expression in E. coli and a western blot was carried out using an anti-His antibody (E). In a DNA ligation assay (F), linearised vector DNA was incubated with either purified LigIV_his, empty vector control (EV) eluate or H2O and used for transformation of E. coli either immediately (0h) or after one hour (1h). After an overnight incubation, surviving clones were counted.

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Figure 3. The ligIV targeting antisense vector pPha-T1_AS (A) was used to create the knock-down lines NAS3 and NAS4 (pfcpA = promoter of fcpA, tfcpA = terminator of fcpA, ble = zeocin resistance gene, bla = ampicillin resistance gene, tfcpa* = shortened version of tfcpA). (B) ligIV mRNA level in wild type (WT) and ligIV knock-down mutants NAS3 and NAS4 were measured in duplex PCR reactions using h4 as calibrator with three biological and three technical replicates per cell line. Data represent means and standard deviations, and statistical confidence level was calculated by one-sided t-test. p < 0.0001 is depicted as four asterisks, whereas three asterisks represent p < 0.001. (C) As one physiological parameter, the photosystem II quantum yield, FV/Fmax, was determined. Data represent means and standard deviations of three technical replicates per each cell line of wild type and knock-down mutants. No significant differences could be determined by one-sided t-test between all cell lines. (D) Survival rate of cells of WT, NAS3 and NAS4 after 5 minutes UV-radiation (1.5 2

Joule/cm ) on plates (+UV (5 min)) was calculated by counting clones after six weeks in comparison to untreated plates (-UV, 100 %).

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Figure 4. Analysis of DNA integration after transformation of wild type shown by southern blots. Sections (modified contrast) of original southern blot images (see suppl. Fig S4) are depicted. (A) Integrated antisense constructs (pPha-T1_AS, see Fig. S2) in knock-down lines NAS3 and NAS4 were detected by probing against ble (zeocin resistance gene). Genomic DNA for the blot was digested with SalI. (B) Integrated HRL constructs (see Fig. S2) in mutants A, B and C were detected by probing against nat (nourseothricin resistance gene). Digestion of genomic DNA was done using NsiI.

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Figure 5. (A) The construct for the HR approach (HRL) on target ligIV is depicted: the nourseothricin resistance gene (nat) is under control of the promoter and terminator of fcpA (pfcpA and tfcpA). This expression cassette is flanked by 1 kb regions (5’F and 3’F) of ligIV (see Fig. 1) and the ampicillin resistance gene bla is shown. In (B), the HRL vector and possible targets for HR by HRL are shown: target ligIV contains homologies to the coding region of ligIV (total homology: 2002 bp), target fcpA contains homologies due to pfcpA and tfcpA (total homology: 705 bp) and the vector backbone of HRL can also target the vector backbone of the antisense construct (total homology: 3116 bp, if a complete integration took place).

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Figure 6. Integration of HRL in ligIV knock-down mutants NAS3 and NAS4 demonstrated by southern blots. Sections (modified contrast) of original southern blot images (see suppl. Fig S4) are depicted. Clones 3A-E were derived from NAS3, whereas clones 4A and 4B resulted from transformation of NAS4. (A) DNA was digested using NsiI. The blot was first probed with nat, a probe against the antibiotic resistance introduced when using HRL. In (B), a comparison of fragments detected on the blot using NsiI for digestion (see Fig. 6A) by probes nat, 3’ligIV, and ble is shown for clone 4B. 3’ligIV detects the 3’ region of ligIV outside the target given by flanking regions, whereas ble probes for the antibiotic resistance introduced by creating the knock-down mutants. For further analysis, DNA was digested with SalI and probed against ble (C), shown in the upper panel. The middle panel shows the results of re-probing again the same blot with fcpA, a probe against the 5’ region prior to the original promotor of fcpA. In the lowest panel, the blot was re-probed against the putative promoter of ligIV (pligIV). The analysis by southern blot of subclones derived from clone 4B is shown in (D), restriction of genomic DNA was done using NsiI. Integrated HRL constructs in clones 4B1 and 4B2 were detected by probing against nat.

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Figure 7. (A) Schematic depiction of a recovered plasmid (compare Fig. S7A, clone 1) from clone 4B1 using NsiI showing an integration of HRL into an antisense construct with an elimination of the elements ble and tfcpA* from the original construct pPha-T1_AS (compare Fig. 3A) based on recombination between the vector backbones. In (B) another recovered plasmid from clone 4B1 (compare Fig. S7A, clone 2) using NsiI is depicted that consists only of HRL without further genomic parts. (C) The inverted integration of nat from HRL into an antisense construct in subclone 4B1 (compare Fig. 6D, S6A) as demonstrated by sequencing is shown as schematic representation. Also in (D), a schematic representation shows the tandem integration identified by sequencing of two complete HRL copies at the genomic location on chromosome 18 (between bp 308200 and 308213) in clone 3C. The NsiI and MscI restriction sites and resulting fragment sizes are depicted as well. Analysis of the ligIV mRNA level in wild type (WT), ligIV knock-down mutant NAS3 and clone 3C by duplex PCR using h4 as calibrator is shown in (E). Data is given as means and standard deviations of three biological and technical replicates per each cell line. Statistical confidence level was calculated by one-sided t-test and is shown as four asterisks for p < 0.0001.

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Figure 8. (A) The HR target tgl1 is shown as a true to scale schematic representation. The flanking regions ptgl1 (5’ flanking region, putative promoter of tgl1), 3FT (3’ flanking region inside the coding region of tgl1) and ttgl1 (3’ flanking region, putative terminator of tgl1) are shown as well as 5’ and 3’ up-/downstream regions (5T and 3T). (B) The expression cassettes HRT1 and HRT2 were used to transform P. tricornutum for HR on target tgl1. The binding sites for primers used in PCR analysis are shown as connected arrows, the size of expected products is given in brackets.



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Figure 9. PCR and phenotypical analysis of clones obtained by transformation of NAS3 and NAS4 with expression cassette HRT2 (compare Fig. 8). (A) The result of PCR amplification of the tgl1 locus of wild type (WT), NAS3, NAS4, clones T3B1 (derived from NAS3) and T4B1 (derived from NAS4) is shown. T3B1 and T4B1 displayed an additional fragment at around 6.5 kb compared to WT, NAS3 and NAS4. These fragments were extracted, purified and the eluate was used as template for nested PCR (B) testing for nat and the tgl1 locus marker 3T. (C) For clone T4B1, sequencing revealed a multiple integration of the elements nat and 3FT (orientation shown by arrows) in complete and incomplete versions, indicated by the length of elements. Phenotypical analysis of the expected triacylglycerol accumulation in T3B1 and T4B1 was done by Nile Red staining and subsequent fluorescence analysis: The result of fluorescence spectroscopy of cells of WT, NAS3, NAS4, T3B1 and T4B1 is shown in (D). Data is given as means and standard deviations of three technical replicates per each cell line. Statistical confidence level was calculated by one-sided t-test and is shown as two asterisks for p < 0.01 and four asterisks for p < 0.0001. Pictures from fluorescence microscopy of selected cells (for an overview of cells see suppl. Fig. S9) after 3D reconstruction of z-stacks and applied top-views are shown in (E): Nile Red fluorescence (yellow) in living cells (chlorophyll autofluorescence, red) was recorded for WT, T3B1 and T4B1.

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Graphical abstract

ACS Paragon Plus Environment

Knock-down of a ligIV homolog enables DNA integration via

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