Editing Diatom (section)

==Genetics==
[[File:Phaeodactylum tricornutum.png|thumb|210px| {{center|''[[Phaeodactylum tricornutum]]'' is widely used as a [[model organism]]}}]]

=== Expressed sequence tagging ===
In 2002, the first insights into the properties of the ''[[Phaeodactylum tricornutum]]'' gene repertoire were described using 1,000 [[expressed sequence tag]]s (ESTs).<ref name="Scala 2002">{{cite journal |doi=10.1104/pp.010713 |pmid=12114555 |pmc=166495 |title=Genome Properties of the Diatom Phaeodactylum tricornutum |journal=Plant Physiology |volume=129 |issue=3 |pages=993–1002 |year=2002 |last1=Scala |first1=S. |last2=Carels |first2=N |last3=Falciatore |first3=A |last4=Chiusano |first4=M. L. |last5=Bowler |first5=C }}</ref> Subsequently, the number of ESTs was extended to 12,000 and the diatom EST database was constructed for functional analyses.<ref name="Maheswari 2005">{{cite journal |doi=10.1093/nar/gki121 |pmid=15608213 |pmc=540075 |title=The Diatom EST Database |journal=Nucleic Acids Research |volume=33 |issue=Database issue |pages=D344–7 |year=2004 |last1=Maheswari |first1=U. |last2=Montsant |first2=A |last3=Goll |first3=J |last4=Krishnasamy |first4=S |last5=Rajyashri |first5=K. R. |last6=Patell |first6=V. M. |last7=Bowler |first7=C. }}</ref> These sequences have been used to make a comparative analysis between ''P. tricornutum'' and the putative complete proteomes from the [[green algae|green alga]] ''[[Chlamydomonas reinhardtii]]'', the [[red alga]] ''[[Cyanidioschyzon merolae]]'', and the diatom ''[[Thalassiosira pseudonana]]''.<ref name="Montsant 2005">{{cite journal |doi=10.1104/pp.104.052829 |pmid=15665249 |pmc=1065351 |title=Comparative Genomics of the Pennate Diatom Phaeodactylum tricornutum |journal=Plant Physiology |volume=137 |issue=2 |pages=500–13 |year=2005 |last1=Montsant |first1=A. |last2=Jabbari |first2=K |last3=Maheswari |first3=U |last4=Bowler |first4=C }}</ref> The diatom EST database now consists of over 200,000 ESTs from ''P. tricornutum'' (16 libraries) and ''T. pseudonana'' (7 libraries) cells grown in a range of different conditions, many of which correspond to different abiotic stresses.<ref>{{cite journal |doi=10.1093/nar/gkn905 |pmid=19029140 |pmc=2686495 |title=Update of the Diatom EST Database: A new tool for digital transcriptomics |journal=Nucleic Acids Research |volume=37 |issue=Database issue |pages=D1001–5 |year=2009 |last1=Maheswari |first1=U. |last2=Mock |first2=T. |last3=Armbrust |first3=E. V. |last4=Bowler |first4=C. }}</ref>

=== Genome sequencing ===
[[File:Thalassiosira pseudonana.jpg|thumb|210px| {{center|''[[Thalassiosira pseudonana]]'' was the first eukaryotic marine phytoplankton to  have its genome sequenced}}]]

In 2004, the entire [[genome]] of the centric diatom, ''[[Thalassiosira pseudonana]]'' (32.4 Mb) was sequenced,<ref name="Armbrust 2004">{{cite journal |doi=10.1126/science.1101156 |pmid=15459382 |title=The Genome of the Diatom Thalassiosira Pseudonana: Ecology, Evolution, and Metabolism |journal=Science |volume=306 |issue=5693 |pages=79–86 |year=2004 |last1=Armbrust |first1=E. V. |last2=Berges |first2=John A. |last3=Bowler |first3=Chris |last4=Green |first4=Beverley R. |last5=Martinez |first5=Diego |last6=Putnam |first6=Nicholas H. |last7=Zhou |first7=Shiguo |last8=Allen |first8=Andrew E. |last9=Apt |first9=Kirk E. |last10=Bechner |first10=Michael |last11=Brzezinski |first11=Mark A. |last12=Chaal |first12=Balbir K. |last13=Chiovitti |first13=Anthony |last14=Davis |first14=Aubrey K. |last15=Demarest |first15=Mark S. |last16=Detter |first16=J. Chris |last17=Glavina |first17=Tijana |last18=Goodstein |first18=David |last19=Hadi |first19=Masood Z. |last20=Hellsten |first20=Uffe |last21=Hildebrand |first21=Mark |last22=Jenkins |first22=Bethany D. |last23=Jurka |first23=Jerzy |last24=Kapitonov |first24=Vladimir V. |last25=Kröger |first25=Nils |last26=Lau |first26=Winnie W. Y. |last27=Lane |first27=Todd W. |last28=Larimer |first28=Frank W. |last29=Lippmeier |first29=J. Casey |last30=Lucas |first30=Susan | display-authors = 6 | bibcode=2004Sci...306...79A |citeseerx=10.1.1.690.4884 |s2cid=8593895 }}</ref> followed in 2008 with the sequencing of the pennate diatom, ''[[Phaeodactylum tricornutum]]'' (27.4 Mb).<ref name="Bowler 2008">{{cite journal |doi=10.1038/nature07410 |pmid=18923393 |title=The Phaeodactylum genome reveals the evolutionary history of diatom genomes |journal=Nature |volume=456 |issue=7219 |pages=239–244 |year=2008 |last1=Bowler |first1=Chris |last2=Allen |first2=Andrew E. |last3=Badger |first3=Jonathan H. |last4=Grimwood |first4=Jane |last5=Jabbari |first5=Kamel |last6=Kuo |first6=Alan |last7=Maheswari |first7=Uma |last8=Martens |first8=Cindy |last9=Maumus |first9=Florian |last10=Otillar |first10=Robert P. |last11=Rayko |first11=Edda |last12=Salamov |first12=Asaf |last13=Vandepoele |first13=Klaas |last14=Beszteri |first14=Bank |last15=Gruber |first15=Ansgar |last16=Heijde |first16=Marc |last17=Katinka |first17=Michael |last18=Mock |first18=Thomas |last19=Valentin |first19=Klaus |last20=Verret |first20=Fréderic |last21=Berges |first21=John A. |last22=Brownlee |first22=Colin |last23=Cadoret |first23=Jean-Paul |last24=Chiovitti |first24=Anthony |last25=Choi |first25=Chang Jae |last26=Coesel |first26=Sacha |last27=De Martino |first27=Alessandra |last28=Detter |first28=J. Chris |last29=Durkin |first29=Colleen |last30=Falciatore |first30=Angela |display-authors = 6 | bibcode=2008Natur.456..239B |s2cid=4415177 |doi-access=free }}</ref> Comparisons of the two reveal that the ''P. tricornutum'' genome includes fewer genes (10,402 opposed to 11,776) than ''T. pseudonana''; no major synteny (gene order) could be detected between the two genomes. ''T. pseudonana'' genes show an average of ~1.52 introns per gene as opposed to 0.79 in ''P. tricornutum'', suggesting recent widespread intron gain in the centric diatom.<ref name="Bowler 2008" /><ref>{{cite journal |doi=10.1093/molbev/msm048 |pmid=17350938 |title=A Very High Fraction of Unique Intron Positions in the Intron-Rich Diatom Thalassiosira pseudonana Indicates Widespread Intron Gain |journal=Molecular Biology and Evolution |volume=24 |issue=7 |pages=1447–57 |year=2007 |last1=Roy |first1=S. W. |last2=Penny |first2=D. |doi-access=free }}</ref> Despite relatively recent evolutionary divergence (90 million years), the extent of molecular divergence between centrics and pennates indicates rapid evolutionary rates within the [[Bacillariophyceae]] compared to other [[eukaryotic]] groups.<ref name="Bowler 2008" /> [[Comparative genomics]] also established that a specific class of [[transposable elements]], the Diatom Copia-like retrotransposons (or CoDis), has been significantly amplified in the ''P. tricornutum'' genome with respect to ''T. pseudonana'', constituting 5.8 and 1% of the respective genomes.<ref name="Maumus etal 2009">{{cite journal |doi=10.1186/1471-2164-10-624 |pmid=20028555 |pmc=2806351 |title=Potential impact of stress activated retrotransposons on genome evolution in a marine diatom |journal=BMC Genomics |volume=10 |pages=624 |year=2009 |last1=Maumus |first1=Florian |last2=Allen |first2=Andrew E |last3=Mhiri |first3=Corinne |last4=Hu |first4=Hanhua |last5=Jabbari |first5=Kamel |last6=Vardi |first6=Assaf |last7=Grandbastien |first7=Marie-Angèle |last8=Bowler |first8=Chris |doi-access=free }}</ref>

===Endosymbiotic gene transfer===
Diatom genomics brought much information about the extent and dynamics of the endosymbiotic [[gene transfer]] (EGT) process. Comparison of the ''T. pseudonana'' proteins with homologs in other organisms suggested that hundreds have their closest homologs in the Plantae lineage. EGT towards diatom genomes can be illustrated by the fact that the ''T. pseudonana'' genome encodes six proteins which are most closely related to genes encoded by the ''[[Guillardia theta]]'' ([[cryptomonad]]) [[nucleomorph]] genome. Four of these genes are also found in red algal plastid genomes, thus demonstrating successive EGT from red algal plastid to red algal nucleus (nucleomorph) to heterokont host nucleus.<ref name="Armbrust 2004" /> More recent [[phylogenomics|phylogenomic analyses]] of diatom proteomes provided evidence for a [[prasinophyte]]-like endosymbiont in the common ancestor of [[chromalveolates]] as supported by the fact the 70% of diatom genes of Plantae origin are of green lineage provenance and that such genes are also found in the genome of other [[stramenopile]]s. Therefore, it was proposed that chromalveolates are the product of serial secondary [[endosymbiosis]] first with a [[green algae]], followed by a second one with a [[red algae]] that conserved the genomic footprints of the previous but displaced the green plastid.<ref>{{cite journal |doi=10.1126/science.1172983 |pmid=19556510 |title=Genomic Footprints of a Cryptic Plastid Endosymbiosis in Diatoms |journal=Science |volume=324 |issue=5935 |pages=1724–6 |year=2009 |last1=Moustafa |first1=A. |last2=Beszteri |first2=B. |last3=Maier |first3=U. G. |last4=Bowler |first4=C. |last5=Valentin |first5=K. |last6=Bhattacharya |first6=D. |bibcode=2009Sci...324.1724M |s2cid=11408339 |url=http://epic.awi.de/20816/1/Mou2009a.pdf |access-date=13 January 2019 |archive-url=https://web.archive.org/web/20140421015051/http://epic.awi.de/20816/1/Mou2009a.pdf |archive-date=21 April 2014 |url-status=dead }}</ref> However, phylogenomic analyses of diatom proteomes and chromalveolate evolutionary history will likely take advantage of complementary genomic data from under-sequenced lineages such as red algae.

===Horizontal gene transfer===
In addition to EGT, [[horizontal gene transfer]] (HGT) can occur independently of an endosymbiotic event. The publication of the ''P. tricornutum'' genome reported that at least 587 ''P. tricornutum'' genes appear to be most closely related to bacterial genes, accounting for more than 5% of the ''P. tricornutum'' proteome. About half of these are also found in the ''T. pseudonana'' genome, attesting their ancient incorporation in the diatom lineage.<ref name="Bowler 2008" />

===Genetic engineering===
To understand the biological mechanisms which underlie the great importance of diatoms in geochemical cycles, scientists have used the ''[[Phaeodactylum tricornutum]]'' and ''[[Thalassiosira]] spp.'' species as model organisms since the 90's.<ref name="genome-editing">{{cite journal |display-authors=2 |last1=Kroth |first1=Peter G. |last2=Bones |first2=Atle M. |last3=Daboussi |first3=Fayza |title=Genome editing in diatoms: achievements and goals |journal=Plant Cell Reports |date=Oct 2018 |volume=37 |issue=10 |pages=1401–1408 |doi=10.1007/s00299-018-2334-1 |pmid=30167805 |bibcode=2018PCelR..37.1401K |s2cid=52133809 |url=http://nbn-resolving.de/urn:nbn:de:bsz:352-2-1lxrzq3xck4wo8 |access-date=20 May 2021 |archive-date=19 February 2022 |archive-url=https://web.archive.org/web/20220219150345/https://kops.uni-konstanz.de/handle/123456789/44586 |url-status=live |hdl=11250/2590892 |hdl-access=free }}</ref>
Few molecular biology tools are currently available to generate mutants or transgenic lines : [[plasmids]] containing transgenes are inserted into the cells using the [[biolistic]] method<ref name="biolistic">{{cite journal |display-authors=2 |last1=Falciatore |first1=Angela |last2=Casotti |first2=Raffaella |last3=Leblanc |first3=Catherine |last4=Abrascia |first4=Chiara |last5=Bowler |first5=Chris |title=Transformation of Nonselectablporter Genes in Marine Diatomse Re |journal=Marine Biotechnology |date=May 2015 |volume=1 |issue=3 |pages=239–251 |doi=10.1007/PL00011773|pmid=10383998 |s2cid=22267097 }}</ref> or transkingdom [[bacterial conjugation]]<ref name="conjugation">{{cite journal |last1=Karas |first1=Bogumil J. |last2=Diner |first2=Rachel E. |last3=Lefebvre |first3=Stephane C. |last4=McQuaid |first4=Jeff |last5=Phillips |first5=Alex P. R. |last6=Noddings |first6=Chari M. |last7=Brunson |first7=John K. |last8=Valas |first8=Ruben E. |last9=Deerinck |first9=Thomas J. |last10=Jablanovic |first10=Jelena |last11=Gillard |first11=Jeroen T. F. |last12=Beeri |first12=Karen |last13=Ellisman |first13=Mark H. |last14=Glass |first14=John I. |last15=Hutchison Iii |first15=Clyde A. |last16=Smith |first16=Hamilton O. |last17=Venter |first17=J. Craig |last18=Allen |first18=Andrew E. |last19=Dupont |first19=Christopher L. |last20=Weyman |first20=Philip D. | display-authors = 6 | title=Designer diatom episomes delivered by bacterial conjugation |journal=Nature Communications |date=21 April 2015 |volume=6 |issue=1 |page=6925 |doi=10.1038/ncomms7925 |pmid=25897682 |pmc=4411287 |bibcode=2015NatCo...6.6925K |language=en |issn=2041-1723|doi-access=free }}</ref> (with 10<sup>−6</sup> and 10<sup>−4</sup> yield respectively<ref name="biolistic" /><ref name="conjugation" />), and other classical transfection methods such as [[electroporation]] or use of [[Polyethylene glycol|PEG]] have been reported to provide results with lower efficiencies.<ref name="conjugation" />

Transfected plasmids can be either randomly integrated into the diatom's chromosomes or maintained as stable circular [[episomes]] (thanks to the CEN6-ARSH4-HIS3 yeast centromeric sequence<ref name="conjugation" />). The phleomycin/[[Zeocin|zeocin resistance gene Sh Ble]] is commonly used as a selection marker,<ref name="genome-editing" /><ref name="slattery">{{cite journal |last1=Slattery |first1=Samuel S. |last2=Diamond |first2=Andrew |last3=Wang |first3=Helen |last4=Therrien |first4=Jasmine A. |last5=Lant |first5=Jeremy T. |last6=Jazey |first6=Teah |last7=Lee |first7=Kyle |last8=Klassen |first8=Zachary |last9=Desgagné-Penix |first9=Isabel |last10=Karas |first10=Bogumil J. |last11=Edgell |first11=David R. | display-authors = 6 | title=An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum |journal=ACS Synthetic Biology |date=16 February 2018 |volume=7 |issue=2 |pages=328–338 |doi=10.1021/acssynbio.7b00191|pmid=29298053 }}</ref> and various transgenes have been successfully introduced and expressed in diatoms with stable transmissions through generations,<ref name="conjugation" /><ref name="slattery" /> or with the possibility to remove it.<ref name="slattery" />

Furthermore, these systems now allow the use of the [[CRISPR gene editing|CRISPR-Cas genome edition tool]], leading to a fast production of functional [[Gene knockout|knock-out mutants]]<ref name="slattery" /><ref name="CRISPR">{{cite journal |display-authors=2 |last1=Nymark |first1=Marianne |last2=Sharma |first2=Amit Kumar |last3=Sparstad |first3=Torfinn |last4=Bones |first4=Atle M. |last5=Winge |first5=Per |title=A CRISPR/Cas9 system adapted for gene editing in marine algae |journal=Scientific Reports |date=July 2016 |volume=6 |issue=1 |pages=24951 |doi=10.1038/srep24951|pmid=27108533 |pmc=4842962 |bibcode=2016NatSR...624951N |doi-access=free }}</ref> and a more accurate comprehension of the diatoms' cellular processes.