Name: silencing the spark: crispr/cas9 genome editing in weakly electric fish
Uploaded: 2019-10-27T14:00:00
Description: Watch this Scientific Journal Video about Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish at JoVE.com

Forfatterne anerkender den heroiske indsats af Monica Lucas, Katherine Shaw, Ryan Taylor, Jared Thompson, Nicole Robichaud, og Hope Healey for at få hjælp til fiskeopdræt, dataindsamling og tidlig protokol udvikling. Vi vil også gerne takke de tre anmeldere for deres forslag til manuskriptet. Vi mener, at det endelige produkt er af bedre kvalitet efter at have rettet deres kommentarer. Dette arbejde blev finansieret af støtte fra National Science Foundation #1644965 og #1455405 til JRG, og Generaldirektoratet for naturvidenskab og ingeniør forskning giver VLS.

Alle metoder, der er beskrevet her, er blevet godkendt af det institutionelle udvalg for dyrepasning og-brug (IACUC) på Michigan State University.
1. valg af sgRNA-mål
Bemærk: Der findes en protokol til manuel design af sgRNAs i trin 1,1. Dette blev udnyttet til scn4aa Target udvælgelse. Der gives en tillægsprotokol for at lette denne proces (trin 1,2) ved hjælp af webportalen EFISHGENOMICS. Det tilrådes, at brugerne vælger protokol 1...

Den fænotypiske rigdom af svagt elektriske fisk, sammen med en nylig spredning af genomforskning ressourcer, motiverer et stærkt behov for funktionelle genomiske værktøjer i svagt elektrisk fisk model. Dette system er særligt attraktivt på grund af den konvergerende udvikling af talrige fænotypiske træk i parallelle nedstamningens af fisk, som let holdes i laboratoriet.
Den protokol, der er beskrevet her, viser effekten af crispr/Cas9-teknikken i nedstamningens af svagt elektriske fisk...

Electroreception og electrogenesis har ændret sig i den evolutionære historie af hvirveldyr. To nedstamningens af teleost fisk, osteoglossiformes og Siluriformes, udviklet electroreception parallelt, og fem nedstamningens af teleoster (gymnotiformes, mormyrids, og slof astroscopus, malapterurus, og Synodontis) udviklet electrogenesis parallelt. Der er en slående grad af konvergens i disse selvstændigt afledte fænotyper, som deler en fælles genetisk arkitektur1,2,3.
Dette er måske bedst eksemplificeret ved de talrige konvergerende funktioner i gymnotiforms og mormyrids, to artsrige teleost clades, som producerer og detekterer svage elektriske felter og kaldes svagt elektriske fisk. I 50 år siden opdagelsen af, at svagt elektriske fisk bruger elektricitet til at fornemme deres omgivelser og kommunikere4, et voksende samfund af videnskabsfolk har fået enorm indsigt i udviklingen af udviklingen1,5 ,6, systemer og kredsløb neurovidenskab7,8,9,10, cellulære fysiologi11,12, økologi og energetics13 ,14,15,16,17, opførsel18,19og makroevolution3,20,21 .
For nylig har der været en spredning af genomisk, transkriptomic og proteomiske ressourcer til elektriske fisk1,22,23,24,25,26, 27,28. Brugen af disse ressourcer har allerede givet vigtig indsigt i forbindelsen mellem genotype og fænotype i disse arter1,2,3,28,29 ,30. En væsentlig hindring for at integrere genomforskning-data med fænotypiske data om svagt elektriske fisk er en nuværende mangel på funktionelle genomforskning-værktøjer31.
Et sådant værktøj er den nyligt udviklede klynge regelmæssigt interspaced korte palindromic gentagelser parret med Cas9 endonuklease (crispr/Cas9, crispr) teknik. Crispr/Cas9 er et genomredigerings værktøj, der er blevet udbredt i både model32,33,34 og ikke-modelorganismer35,36,37 ens. CRISPR/Cas9-teknologien har udviklet sig til et punkt, hvor et laboratorium, der er i stand til at grundlæggende molekylær biologi, nemt kan generere gen-specifikke sonder kaldet Short guide RNAs (sgRNAs) til en lav pris ved hjælp af en ikke-klonings metode38. Crispr har fordele i forhold til andre knockout/Knockdown strategier, såsom morpholinos39,40, transskription aktivator-lignende Effector nucleaser (talens), og zink finger nucleaser (zfns), som er dyre og tidskrævende at generere for hvert målgen.
CRISPR/Cas9-systemet fungerer til at skabe genudskæringer ved at målrette mod et bestemt område af genomet, instrueret af sgRNA-sekvensen, og forårsage en dobbeltstrenget pause. Den dobbeltstrengede pause detekteres af cellen og udløser endogene DNA-reparationsmekanismer fortrinsvis ved hjælp af NHEJ-vejen (non-homologous end-forbindelse). Denne vej er meget fejlbehæftet: under reparationsprocessen vil DNA-molekylet ofte indeholde indsættelser eller sletninger på det dobbelt strandede Break site. Disse indeler kan resultere i tab af funktion på grund af enten (1) Skift i den åbne læse ramme, (2) indsættelse af en tidlig stop codon, eller (3) forskydninger i den kritiske primære struktur af genproduktet. I denne protokol udnytter vi CRISPR/Cas9 editing til at målrette punktmutationer i målgener ved hjælp af NHEJ i svagt elektriske fiskearter. Selv om denne mutagenicese metode er enklere og mere effektiv end andre teknikker, forventes den at resultere i en række fænotypiske severiteter i F0, som tilskrives genetisk mosaik41,42,43 ,44.
Udvælgelse af organismer Med henblik på at lette fremtidige undersøgelser af den sammenlignende genomforskning i svagt elfisk er der behov for at udvalgte en repræsentativ dyreart for både gymnotiforme og mormyrier til protokol udvikling. Efter drøftelserne under 2016 Electric Fish-mødet i Montevideo i Uruguay var der enighed i Fællesskabet om at anvende arter, der allerede kunne opdrættes i laboratoriet, og som havde genøkonomiske ressourcer til rådighed. Gymnotiformen Brachyhypopomus gauderio og mormyrid Brienomyrus brachyistius blev udvalgt som arter, der passede til disse kriterier. I begge arter er naturlige signaler til at fremkalde og vedligeholde avlsforhold let at efterligne i fangenskab. B. gauderio, en nøgterne arter fra Sydamerika, har den fordel, at lave opdræts krav: fisk kan holdes på relativt høj tæthed i relativt små (4 L) tanke. B. gauderio har også en hurtig generations omsætning under bundne forhold. Under laboratorieforhold kan B. gauderio udvikle sig fra æg til voksen i ca. 4 måneder.
B. brachyistius, en art af mormyrid fisk fra vest-central Afrika, racer let i fangenskab. B. brachyistius er let tilgængelig gennem akvariet handel, har været meget udbredt i mange undersøgelser, og nu har en række genøkonomiske ressourcer til rådighed. Deres livscyklus strækker sig over 1 − 3 år afhængigt af laboratorieforholdene. Opdræts kravene er noget mere intensive for denne art, der kræver moderat store tanke (50 − 100 L) på grund af deres aggression under avl.
Laboratorier, der studerer andre arter af elektriske fisk, bør let kunne tilpasse denne protokol, så længe arterne kan opdrættes, og enkelt celle embryoner kan opsamles og opdrættes til voksenalderen. Boliger, larve opdræt og in vitro fertilisering (IVF) satser vil sandsynligvis ændre sig med andre arter; denne protokol kan dog bruges som udgangspunkt for Avlsforsøg i andre svagt elektriske fisk.
Et ideelt Genmål for bevis for koncept: scn4aa Svagt elektrisk mormyrid og gymnotiform fisk genererer elektriske felter (electrogenesis) ved at aflade et specialiseret organ, kaldet el-orgel. Elektriske organ udledninger (EODs) skyldes samtidig fremstilling af aktionspotentialer i de elektriske organ celler kaldet elektro cytter. EODs detekteres af en række elektroreceptorer i huden for at skabe elektriske billeder i høj opløsning af fiskens omgivelser45. Svagt elektriske fisk er også i stand til at afsløre funktioner i deres konspecificitet ' EOD bølgeformer18 samt deres udledning satser46, tillader EODS at fungere yderligere som et socialt kommunikations signal analogt med fuglesang eller frø vokaliseringer47.
En hovedkomponent i action potentiel generation i elektro cytterne af både mormyrid og gymnotiform svagt elektriske fisk er den spændings-gated natrium kanal NaV 1.42. Ikke-elektriske teleoster udtrykker to paralogous genkopier, scn4aa og scn4ab, som koder for den spændings-gated natrium kanal NAV 1.430. I både gymnotiform og mormyrid svagt elektrisk fisk lineages, scn4aa har udviklet sig hurtigt og undergået talrige aminosyre substitutioner, der påvirker dens kinetiske egenskaber48. Vigtigst er, scn4aa er blevet opdelt i begge nedstamningens til elorgel2,3. Det relativt begrænsede udtryk for scn4aa til det elektriske orgel, samt dets nøglerolle i genereringen af EODS, gør det til et ideelt mål for crispr/Cas9 knockout eksperimenter, da det har minimal skadelige pleiotropic effekter. Fordi svagt elektriske fisk begynder at aflade deres larve elektriske organer 6 − 8 dage efter befrugtning (DPF), målretning af scn4aa er ideelt egnet til hurtig fænotypebestemmelse efter embryo mikroinjektion.

<table>
	<tbody>
		<tr>
			<td>20 mg/mL RNA grade Glycogen</td>
			<td>Thermo Scientific</td>
			<td>R0551</td>
			<td></td>
		</tr>
		<tr>
			<td>50 bp DNA ladder</td>
			<td>NEB</td>
			<td>N3236L</td>
			<td></td>
		</tr>
		<tr>
			<td>borosilicate glass capillary with filament</td>
			<td>Sutter Instrument</td>
			<td>BF100-58-10</td>
			<td>(O.D. 1.0mm, I.D. 0.58 mm, 10 cm length)</td>
		</tr>
		<tr>
			<td>Cas9 protein with NLS; 1 mg/mL</td>
			<td>PNA Biology</td>
			<td>CP01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dneasy Blood &#38; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf FemptoJet 4i Microinjector</td>
			<td>Fisher Scientific</td>
			<td>E5252000021</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Microloader Pipette Tips</td>
			<td>Fisher Scientific</td>
			<td>10289651</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Fisher Scientific</td>
			<td>14-824-654</td>
			<td>referred to as &#34;precision glass syringe&#34; in the protocol</td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Fisher Scientific</td>
			<td>06-666</td>
			<td>referred to as &#34;delicate task wipe&#34; in the protocol</td>
		</tr>
		<tr>
			<td>MEGAscript T7 Transcription Kit</td>
			<td>Invitrogen</td>
			<td>AM1334</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuffer 3</td>
			<td>NEB</td>
			<td>B7003S</td>
			<td>used for in vitro cleavage assay</td>
		</tr>
		<tr>
			<td>OneTaq DNA kit</td>
			<td>NEB</td>
			<td>M0480L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovaprim</td>
			<td>Syndel USA</td>
			<td>https://www.syndel.com/ovaprim-ovammmlu010.html</td>
			<td>referred to as &#34;spawning agent&#34; in the protocol</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>S37440</td>
			<td>referred to as &#34;thermoplastic&#34; in the protocol</td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>WPI</td>
			<td>SU-P97</td>
			<td>sutter brand</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>Reusable needle- requires customization</td>
			<td>Fisher Scientific</td>
			<td>7803-02</td>
			<td>Customize to 0.7 inches long; point style 4 and angle 25</td>
		</tr>
		<tr>
			<td>T4 DNA polymerase</td>
			<td>NEB</td>
			<td>M0203L</td>
			<td>Use with the 10X NEB buffer that is included</td>
		</tr>
		<tr>
			<td>Teflon coated tools</td>
			<td>bonefolder.com</td>
			<td>T-SPATULA4PIECE</td>
			<td>referred to as &#34;polytetrafluoroethene&#34; in the protocol</td>
		</tr>
	</tbody>
</table>

silencing the spark: crispr/cas9 genome editing in weakly electric fish

Electroreception og electrogenesis har ændret sig i den evolutionære historie af hvirveldyr. Der er en slående grad af konvergens i disse selvstændigt afledte fænotyper, som deler en fælles genetisk arkitektur. Dette er måske bedst eksemplificeret ved de talrige konvergerende funktioner i gymnotiforms og mormyrids, to artsrige teleost clader, der producerer og detekterer svage elektriske felter og kaldes svagt elektriske fisk. I 50 år siden opdagelsen af, at svagt elektriske fisk bruger elektricitet til at fornemme deres omgivelser og kommunikere, et voksende samfund af videnskabsfolk har fået enorm indsigt i udviklingen af udvikling, systemer og kredsløb neurovidenskab, cellulære fysiologi, økologi, evolutionær biologi, og opførsel. For nylig har der været en spredning af genomressourcerne til elektriske fisk. Brugen af disse ressourcer har allerede lettet vigtig indsigt med hensyn til forbindelsen mellem genotype og fænotype hos disse arter. En væsentlig hindring for at integrere genomforskning-data med fænotypiske data om svagt elektriske fisk er en nuværende mangel på funktionelle genomforskning-værktøjer. Vi rapporterer her en fuld protokol for at udføre CRISPR/Cas9 mutagenese, der udnytter endogene DNA reparationsmekanismer i svagt elektriske fisk. Vi viser, at denne protokol er lige så effektiv i både mormyrid-arterne Brienomyrus brachyistius og gymnotiformen Brachyhypopomus gauderio ved at bruge crispr/Cas9 til at målrette indels og punktmutationer i den første exon af natrium kanalen gen scn4aa. Ved hjælp af denne protokol blev embryoner fra begge arter fremstillet og Geno skrevet for at bekræfte, at de forventede mutationer i den første exon af natrium kanalen scn4aa var til stede. Knock-out succes fænotype blev bekræftet med optagelser viser reduceret elektrisk organ udledning amplituder sammenlignet med uinjiceret størrelse-matchede kontrol.

SgRNA-målstederne blev identificeret inden for exon 1 af scn4aa i både b. Gauderio og b. brachyistius som beskrevet i afsnit 1. SgRNAs blev genereret som beskrevet i afsnit 2. Efter vellykket sgRNA-udvælgelse og-syntese (figur 1) blev in vitro-spaltning afprøvet (figur 2). SgRNAs, der demonstrerer in vitro-skæring, blev derefter udvalgt til mikroinjektioner med enkelt celle.
Voksne fisk var konditionere...

Watch this Scientific Journal Video about Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish at JoVE.com

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Her præsenteres en protokol for at producere og bageste crispr/Cas9 genom knockout elektrisk fisk. Detaljeret beskrevet er de nødvendige molekylær biologi, avl og opdræts krav for både en gymnotiform og en mormyrid, og injektion teknikker til at producere Cas9-induceret Indel F0 larver.

Lyddæmpnings spark: CRISPR/Cas9 Genome redigering i svagt elektrisk fisk

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

CRISPR/Cas9-genredigering i svagt elektriske fisk, især i en komparativ sammenhæng, vil gøre det muligt for forskere at teste hypoteser om, hvordan specifikke gener og genetisk varians bidrager til fænocypisk variation. CRISPR/Cas9 er en effektiv metode til generering af mutante F-nul embryoner. Denne protokol giver forskerne mulighed for at foretage CRISPR-Cas9 manipulationer i to konvergerende udviklede arter af svagt elektriske fisk.Nøglen til at bruge denne fremgangsmåde med nogle ændringer i andre elektriske fisk eller andre fiskearter er tilgængeligheden af transskriptionologiske eller genomiske ressourcer til at gøre sgRNAs. Det kan være svært at udføre mikroinjektioner og visualisere cellerne på et 3D-sted i et 2D-plan. Få en lille zebrafisk koloni for æg til praksis generelle mikroinjektion teknikker.Demonstration af proceduren med Savvas Constantinou vil være Jared Thompson, en tekniker fra mit laboratorium. Efter opbevaring af fiskearter af interesse under passende yngleforhold, identificere hunfisk, der synes gravid. I B.gauderio, vil den kvindelige har hævede gonader bare caudal til udluftning.I B.brachyistius hunner vil have hævede maver og synes dybt rørige. Efter anæstesiadministration tilsættes gydemidlet til fire gange mængden af DPBS i et PCR-rør med grundig blanding. Løsningen bliver uklar.Brug en pipette til at måle den beregnede dosis og dispensere det fortyndede middel til et stykke termoplastisk, før opløsningen trækkes ind i en præcisionsglassprøjte udstyret med en 28-gauge 19 til 25-milliliter længde affaset nål. Injicer opløsningen i dorsal trunk musklen med en jævn hastighed og lad nålen sidde i to til fire sekunder før fjernelse. Derefter straks placere fisk i frisk systemvand til nyttiggørelse.For B.gauderio sperm indsamling, efter 24 timer, skal du vælge en stor hanfisk og så hurtigt som muligt efter sedation, tør fisken grundigt, især omkring hovedet og Vent. Den tørrede fiskes ventrale side op, forreste til venstre i passende holder, dækket af en MS222 gennemblødt fugtigt køkkenrulle med hovedet så parallelt med bordet som muligt. Umiddelbart lægge pres i caudal til rostral retning med lys klemme straks over gonads mod udluftning og ved hjælp af en mikropipetter med en spids, omhyggeligt indsamle sædceller, da det er presset fra hannen i 50-microliter intervaller.Tilføj aliquot af sædceller direkte på 500-microliter mængder af sperm extender opløsning på is. Umiddelbart efter indsamlingen, placere fisk i slim pels beskyttelse produktbehandlet frisk systemvand. For B.gauderio æg indsamling, efter tørring en bedøvet B.gauderio hun fisk, straks placere fisk på sin side med hovedet vender den ikke-dominerende hånd og anvende pres i caudal til rostral retning med lys klemme medialt over gonads mod udluftning.Ved hjælp af en polytetrafluorethylen værktøj, omhyggeligt indsamle æggene, da de er presset fra cloaca og hurtigt placere æggene i en lille overdækket petriskål. Hvis du arbejder alene efter klemme, røre ægmassen til bunden af den lille petriskål, da de er bortvist fra hunnen. Umiddelbart efter opsamling af æggene, placere fisken i slim coat beskyttelse produktbehandlet frisk systemvand.For B.gauderio in vitro befrugtning tilsættes 100 mikroliter af den godt blandede sæd ses-opløsning direkte over ægmassen efterfulgt af en milliliter frisk systemvand. Bland gametes suspension grundigt i 30 til 60 sekunder, før du tilføjer en ekstra en til to milliliter 0,22-mikrometer pore filtreret system vand til cellerne. Mærk skålen med in vitro-befrugtningstiden, og placer skålen i en 29-graders Celsius-inkubator, der sætter en 50-minutters timer for at kontrollere udviklingens forløbet.I befrugtningsperioden skal du bruge en mikrolæsserpipettespids til at baglæse to mikroliter af injektionsopløsningen af interesse i en mikroindøjelignål og udvise væsken så tæt på spidsen som muligt. Når en enkelt celle er dannet ved dyretang i mindst én celle, overføres skålen under et dissekerende mikroskop, og der anvendes et par fine kraftanger til at bryde nålespidsen i en skrå vinkel mod, hvor nålens konus begynder at udvise stivhed. Kanylens boring skal forblive så lille som muligt, samtidig med at der opretholdes en stiv konus.Identificer de udviklende zygotes under mikroskopet og brug en plastoverførselspipette med en afskåret spids til at placere 10 til 20 æg i så lidt vand som muligt på kanten af glasmikroskopets dias, der er indstillet i den omvendte toppen af petriskålen. Brug en delikat opgave tørre, forsigtigt trykke på diaset for at fjerne det overskydende vand, så æggene til at holde fast på kanten af diaset. Vand vil være onde under diaset og trække æg mod kanten.Der bør være lige nok vand til at holde æggene fugtige og samtidig opretholde lidt stående fugt for at undgå æg bevægelse under injektion. Juster æggene mod diaset lodret, vinkelret på den fortrivte nål, og brug en mikromanipulator til at placere nålen mod chorion. Derefter holde nålen i en ca 45-graders vinkel, indsætte spidsen først i chorion og derefter, i den enkelte celle, indsprøjtning gennem æggeblomme, hvis det er muligt.Fjern eventuelle ødelagte æg under injektionen med fine fak.a.-ber. Når alle æggene er blevet injiceret, skal du bruge en sprøjte flaske 0,22-mikrometer pore filtreret system vand til forsigtigt at skylle æggene fra injektionsstadiet i en ny 100-millimeter diameter Petriskål. Efter en vellykket kort guide RNAs udvælgelse og syntese, in vitro spaltning kan testes for udvælgelse for enkelt celle mikroinjections.Efter PCR oprydning og kloning, i denne repræsentative eksperiment, CRISPR-Cas9 induceret mutationer blev identificeret i individuelle B.gauderio og B.brachyistius kloner med stærk elektrisk organ udledning amplitude reduktion, mens ikke-injected kontrol vises kun refereres genotyper. Visualisering af amplituden for elektriske organer mellem bekræftede CRISPR-mutanter og agentstørrelse matchede ikke-injicerede kontroller viste, at både SCN-4 AA mutant B.brachyistius og B.gauderio embryoner havde betydeligt lavere elektriske organudladnings amplituder end kontroller. Når mikroinjektionerne udføres, skal du bevæge dig så hurtigt og bevidst som muligt for at maksimere antallet af encellede indsprøjtede embryoner, der overlever.Spild ikke tiden med svære æg. Når succesfulde mutanter er blevet identificeret, kan traditionelle avlsmetoder skabe stabile mutant linjer, som eliminerer CRSPR associerede mosaicism bekymringer. Denne protokol vil give forskerne mulighed for at gennemføre sammenlignende genomiske undersøgelser med funktionel validering i to konvergerende udviklede arter af svagt elektriske fisk.

silencing-the-spark-crisprcas9-genome-editing-in-weakly-electric-fish

Research

JoVE Journal

Biology

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

Genome Redigering med CompoZr tilpassede zink finger Nucleaser (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 &#181;m&#160;x 200 &#181;m&#160;x 200 &#181;m using LED illumination, to 5 mm&#160;x 5 mm&#160;x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.

Digital Inline Holografisk Microscopy (DIHM) af svagt-sprednings Emner

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

Generation af genomiske deletioner i pattedyrcellelinjer via CRISPR / Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs. Towards this end, much recent work has focused on identifying candidate genes for the evolution of traits in a variety of species. However, until recently it has been challenging to functionally validate interesting candidate genes. Recently developed tools for genetic engineering make it possible to manipulate specific genes in a wide range of organisms. Application of this technology in evolutionarily relevant organisms will allow for unprecedented insight into the role of candidate genes in evolution. Astyanax mexicanus (A. mexicanus) is a species of fish with both surface-dwelling and cave-dwelling forms. Multiple independent lines of cave-dwelling forms have evolved from ancestral surface fish, which are interfertile with one another and with surface fish, allowing elucidation of the genetic basis of cave traits. A. mexicanus has been used for a number of evolutionary studies, including linkage analysis to identify candidate genes responsible for a number of traits. Thus, A. mexicanus is an ideal system for the application of genome editing to test the role of candidate genes. Here we report a method for using transcription activator-like effector nucleases (TALENs) to mutate genes in surface A. mexicanus. Genome editing using TALENs in A. mexicanus has been utilized to generate mutations in pigmentation genes. This technique can also be utilized to evaluate the role of candidate genes for a number of other traits that have evolved in cave forms of A. mexicanus.

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish.

Genome Redigering i<em&gt; Astyanax mexicanus</em&gt; Brug Transskription Activator-lignende Effector Nucleaser (Talens)

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs.  Towards this end, much recent ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

Forberedelse og injektion af embryoner af Culex-myg til generering af Null-mutationer ved hjælp af CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function&#8212;to produce large amounts of antibodies&#8212;can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

Genomteknik af primære humane B-celler ved hjælp af CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

Pupal- og voksenindsprøjtninger til RNAi- og CRISPR-genredigering i Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&amp;#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

TGF-β medieret endotel til mesenchymal overgang (EndMT) og funktionel vurdering af EndMT-effektorer ved hjælp af CRISPR/Cas9-genredigering

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

Konstruktion af homozygote mutanter af migrerende græshoppe ved hjælp af CRISPR / Cas9-teknologi

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...