High-throughput CRISPR Vector Konstruktion og karakterisering af DNA Ændringer af Generation of Tomato Behårede Roots
Summary
Anvendelse af DNA samling, kan flere CRISPR vektorer konstrueres parallelt i en enkelt kloning reaktion, hvilket gør konstruktionen af et stort antal CRISPR vektorer en enkel opgave. Tomat hårrødder er en glimrende modelsystem til at validere CRISPR vektorer og generere mutant materialer.
Abstract
Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.
Introduction
Evnen til at generere målrettede DNA ændringer med CRISPR / Cas9 har et stort potentiale inden for funktionel genomforskning studier. Der er to komponenter af CRISPR / Cas9 systemet; den Cas9 nuklease, afledt af Staphylococcus pyogenes og en ca. 100-nt guide RNA (gRNA) molekyle, der dirigerer Cas9 til det målrettede DNA site (s) 1. Target anerkendelse bibringes af den første ~ 20 nt af gRNA, som giver mulighed for high-throughput produktion af targeting vektorer 2,3. De fleste organismer, der kan manipuleret, allerede har været med CRISPR / Cas9 teknologi 4,5.
I planter, konstitutive promotorer, såsom CaMV 35S-promotoren, anvendes almindeligvis til at drive ekspressionen af Cas9 nuclease 6. De gRNAs udtrykkes ved anvendelse af de RNA-polymerase III U6 eller U3 promotorer, der begrænser den første base af gRNA til enten et G, for U6, eller A for U3, til effektiv transkription. Imidlertid RNA-polymerase II promoters, der er fri for disse begrænsninger, er også blevet anvendt 7,8.
Forskellige gRNAs inducerer DNA-mutationer med forskellige virkningsgrader, og så det kan være vigtigt først at validere CRISPR vektorer før man investerer i hel-plante transformationer eller oprette omfattende fænotypiske skærme. Transient ekspression af CRISPR-konstruktioner i planter ved anvendelse agroinfiltration for eksempel generelt resulterer i en lavere frekvens af DNA modifikation sammenlignet med stabile planter 6, hvilket gør påvisning af mutationer vanskelige og fænotypiske assays upraktisk med sådanne fremgangsmåder. Såkaldte hårede rødder er en bekvem, alternativt system siden kan genereres et stort antal uafhængige, stabilt transformerede materialer inden for uger, i modsætning til måneder for stabile planter. CRISPR vektorer er meget effektive til at inducere DNA-mutationer i hårrødder 9,10.
DNA samlingsmetoder effektivt ligere DNA-fragmenter indeholdende overlapping ender 11. En væsentlig fordel ved nogle DNA samlingsmetoder er evnen til at inkorporere ssDNA (dvs. oligoer) i den samlede produkter. Da gRNAs kun ~ 20 nt lang og kan gøres nye mål med syntetiserede oligoer, disse DNA samlingsmetoder er velegnede til CRISPR kloning. De her beskrevne protokoller er baseret på P201 serie CRISPR vektorer, som er blevet anvendt i sojabønne 10, poppel 12 og nu tomat. Kloningen præsenteret tilbyder flere fordele i forhold til den aktuelle kloning metode 10. Nemlig, kan fuldt funktionelle vektorer blive frembragt i en enkelt kloning reaktion på en enkelt dag. Vector konstruktion kan også samles til at generere flere CRISPR vektorer i parallel, hvilket yderligere reducerer hands-on tid og materialeomkostninger. Vi præsenterer også en protokol til generering af tomater hårrødder som en effektiv metode til at producere transgene materialer med målrettede gendeletioner. Hårrødder bruges til gyldigspiste CRISPR vektorer og tilvejebringe materiale til efterfølgende eksperimenter.
Protocol
Representative Results
Discussion
Since DNA assembly is used to recombine any overlapping DNA sequences, this cloning method can be applied to any CRISPR vector construction. Most CRISPR cloning schemes use either gene synthesis of the gRNA, type IIS restriction enzymes17,18, or overlap-extension PCR19. Each of these techniques has inherent advantages and disadvantages, but they typically require multiple hands-on cloning steps. The primary advantage of the cloning method presented here is that the entire process occurs in a single,…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Denne forskning blev støttet af National Science Foundation tilskud IOS-1025642 (GBM). Vi takker Maria Harrison til tilvejebringelse af ARqua1 stamme.
Materials
| NEBuilder® (HiFi DNA assembly mix) | New England Biolabs | E5520 | |
| p201N:Cas9 | Addgene | 59175 | The p201H:Cas9 plasmid (59176) is also compatible with the reported overlaps and enzymes. |
| pUC gRNA Shuttle | Addgene | 47024 | |
| SwaI | New England Biolabs | R0604S | |
| SpeI | New England Biolabs | R0133S | |
| Zymo clean and concentrator-5 column purification | Zymo Research | D4003 | |
| NEB Buffer 2.1 | New England Biolabs | B7202S | |
| NEB CutSmart (Buffer 4) | New England Biolabs | B7204S | |
| NEB Buffer 3.1 | New England Biolabs | B7203S | |
| EconoSpin Mini Spin Column (plasmid prep) | Epoch Life Sciences | 1910-050/250 | |
| EcoRV-HF® | New England Biolabs | R3195S | |
| StyI-HF® | New England Biolabs | R3500S | |
| MS Salts + Gamborg Vitamins | Phytotechnology Laboratories | M404 | |
| Phytagel™ (gellan gum) | Sigma Aldrich | P8169 | |
| GA-7 Boxes | Sigma Aldrich | V8505 | |
| Micropore™ surgical tape | 3M | 1535-0 | |
| Timentin® (Ticarcillin/Clavulanic acid) | Various | 0029-6571-26 | |
| Primers 5' -> 3' | |||
| SwaI_MtU6F | GATATTAATCTCTTCGATGAAATTTATGCCTATCTTATATGATCAATGAGG | ||
| MtU6R | AAGCCTACTGGTTCGCTTGAAG | ||
| ScaffoldF | GTTTTAGAGCTAGAAATAGCAAGTT | ||
| SpeI_Scaffold R | GTCATGAATTGTAATACGACTCAAAAAAAAGCACCGACTCGGTG | ||
| StUbi3P218R | ACATGCACCTAATTTCACTAGATGT | ||
| ISceIR | GTGATCGATTACCCTGTTATCCCTAG | Cannot be used for Sanger sequencing since there is a second binding site on the plasmid | |
| UNS1_Scaffold R | GAGAATGGATGCGAGTAATGAAAAAAAGCACCGACTCGGTG | ||
| UNS1_MtU6 F | CATTACTCGCATCCATTCTCATGCCTATCTTATATGATCAATGAGG | ||
| p201R | CGCGCCGAATTCTAGTGATCG | ||
| Bolded sequences denote 20-nt overlaps with linearized p201N:Cas9. | |||
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