通过细菌供体质粒接合转移的方法将Cas9或 TevCas9系统导入三角褐指藻

Helen Wang; Samuel S. Slattery; Bogumil J. Karas; David R. Edgell

doi:10.21769/BioProtoc.2974

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Peer-reviewed

Delivery of the Cas9 or TevCas9 System into Phaeodactylum tricornutum via Conjugation of Plasmids from a Bacterial Donor

通过细菌供体质粒接合转移的方法将Cas9或 TevCas9系统导入三角褐指藻

HW Helen Wang

SS Samuel S. Slattery

BK Bogumil J. Karas

DE David R. Edgell email

发布: 2018年08月20日第8卷第16期 DOI: 10.21769/BioProtoc.2974 浏览次数: 7920

评审: David CisnerosRan ChenAnonymous reviewer(s)

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Abstract

Diatoms are an ecologically important group of eukaryotic microalgae with properties that make them attractive for biotechnological applications such as biofuels, foods, cosmetics and pharmaceuticals. Phaeodactylum tricornutum is a model diatom with defined culture conditions, but routine genetic manipulations are hindered by a lack of simple and robust genetic tools. One obstacle to efficient engineering of P. tricornutum is that the current selection methods for P. tricornutum transformants depend on the use of a limited number of antibiotic resistance genes. An alternative and more cost-effective selection method would be to generate auxotrophic strains of P. tricornutum by knocking out key genes involved in amino acid biosynthesis, and using plasmid-based copies of the biosynthetic genes as selective markers. Previous work on gene knockouts in P. tricornutum used biolistic transformation to deliver CRISPR-Cas9 system into P. tricornutum. Biolistic transformation of non-replicating plasmids can cause undesired damage to P. tricornutum due to random integration of the transformed DNA into the genome. Subsequent curing of edited cells to prevent long-term overexpression of Cas9 is very difficult as there is currently no method to excise integrated plasmids. This protocol adapts a new method to deliver the Cas9 or TevCas9 system into P. tricornutum via conjugation of plasmids from a bacterial donor cell. The process involves: 1) design and insertion of a guideRNA targeting the P. tricornutum urease gene into a TevCas9 expression plasmid that also encodes a conjugative origin of transfer, 2) installation of this plasmid in Escherichia coli containing a plasmid (pTA-Mob) containing the conjugative machinery, 3) transfer of the TevCas9 expression plasmid into P. tricornutum by conjugation, 4) screening of ex-conjugants for urease knockouts using T7 Endonuclease I and phenotypic screening, and 5) curing of the plasmid from edited cells.

Keywords: CRISPR-Cas9 (CRISPR-Cas9)

Conjugation (结合)

Phaeodactylum tricornutum (三角褐指藻)

Auxotroph (营养缺陷型)

Genome editing (基因组编辑)

Diatoms (硅藻)

Background

The CRISPR system is a bacterial immune system that recognizes foreign DNA and leads to the activation of a targeted endonuclease, such as Cas9, which will generate a double strand break (DSB) in the invading DNA (Jinek et al., 2012; Wright et al., 2016). This system has been co-opted for genome editing applications whereby a single chimeric guide RNA (gRNA) can program Cas9 to target genes in model organisms, including in P. tricornutum (Daboussi et al., 2014). However, Cas9 generates blunt end DSB and the length of indels generated upon DNA repair is difficult to predict. Thus, in this study we used a dual endonuclease, TevCas9 to target the urease gene. TevCas9 is a dual endonuclease that is generated through the fusion of the I-TevI nuclease domain to Cas9 via a linker region (Wolfs et al., 2016). TevCas9 creates 33-36 bp deletions with non-compatible DNA ends (Wolfs et al., 2016). A previous study used transcription activator-like effector nucleases (TALENs) delivered by biolistic transformation (Weyman et al., 2015) to generate knockouts of the urease gene in P. tricornutum. To create an efficient system for gene editing in P. tricornutum, we adapted and optimized a plasmid-based system to deliver Cas9 or TevCas9 into P. tricornutum via conjugation from a bacterial donor cell (Karas et al., 2015). The new conjugation-based method to deliver Cas9 or TevCas9 described here is simpler, more efficient, and does not require specialized equipment for biolistic transformation (Slattery et al., 2018).

Materials and Reagents

Pipette tips, 100-1,250 μl (VWR, catalog number: 89079-470 )
Pipette tips, 1-200 μl (VWR, catalog number: 89079-478 )
Pipette tips, 0.1-10 μl (VWR, catalog number: 89079-464 )
1.5 ml Eppendorf tubes (Corning, Axygen^®, catalog number: MCT-150-C )
0.2 ml PCR tubes (VWR, catalog number: 20170-012 )
Parafilm (Bemis, catalog number: PM996 )
50 ml centrifuge tubes (Greiner Bio One, catalog number: 227261 )
Nalgene^TM filters (Thermo Fisher Scientific, catalog number: 595-4520 )
Phaeodactylum tricornutum cells [Culture Collection of Algae and Protozoa (CCAP, UK), catalog number: 1055/1 ] grown in 50 ml Falcon tubes
TransforMax^TMEPI300^TM Chemically competent E. coli cells (Lucigen, catalog number: C300C105 )
pKS diaCas9_sgRNA plasmid (Addgene, catalog number: 74923 )
BsaI-HF restriction endonuclease (New England Biolabs, catalog number: R0535S )
P1 buffer (QIAGEN, catalog number: 19051 )
β-mercaptoethanol (VWR, catalog number: 97064-588 )
Alkaline lysis (P2) buffer (QIAGEN, catalog number: 19052 )
Neutralization (P3) buffer (QIAGEN, catalog number: 19053 )
Isopropanol (Sigma-Aldrich, catalog number: 190764 )
95% (v/v) EtOH (Commercial Alcohols, catalog number: P016EA95 )
T4 DNA ligase with buffer (New England Biolabs, catalog number: M0202S )
2-Propanol-205 (Caledon Laboratories, catalog number: 8601-7-40 )
BSA (Sigma-Aldrich, catalog number: A2058 )
BsaI (New England Biolabs, catalog number: R0535S )
EZ-10 spin column Miniprep Kit (Bio Basic, catalog number: BS614 )
EZ-10 spin column PCR product purification kit (Bio Basic, catalog number: BS363 )
Bacto agar (BD, Bacto^TM, catalog number: 214030 )
Agar A (Bio Basic, catalog number: FB0010 )
Yeast extract (BioShop, catalog number: YEX401 )
Bacteriological tryptone (BioShop, catalog number: TRP402 )
Sodium chloride (NaCl) (Fisher Scientific, Fisher Chemical, catalog number: S271-500 )
AmpliTaq 360 DNA polymerase, 10x buffer, and 25 mM Magnesium Chloride (Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4398828 )
Zeocin^TM (InvivoGen, catalog number: ant-zn-5p )
Ampicillin (BioShop, catalog number: AMP201.100 )
Gentamicin sulfate (Bio Basic, catalog number: GB0217 )
D-Glucose (BioShop, catalog number: GLU501.1 )
Zymolyase 20 T (BioShop, catalog number: ZYM001.1 )
Tris (Fisher Scientific, Fisher BioReagents, catalog number: BP152-5 )
Glycerol (Fisher Scientific, Fisher Chemical, catalog number: G33-1 )
Sodium sulfate (Na₂SO₄) (Fisher Scientific, Fisher Chemical, catalog number: S421-300LB )
Potassium chloride (KCl) (Merck, EMD Millipore, catalog number: PX1405-1 )
Sodium bicarbonate (NaHCO₃) (Merck, EMD Millipore, catalog number: SX0320-1 )
Potassium bromide (KBr) (The Science Company, catalog number: NC-2136 )
Boric acid (H₃BO₃) (BioShop, catalog number: BOR001.1 )
Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S7920 )
Magnesium chloride hexahydrate (MgCl₂•6H₂O) (BioShop, catalog number: MAG510.1 )
Calcium chloride dihydrate (CaCl₂•2H₂O) (Fisher Scientific, catalog number: C79-3 )
Strontium chloride hexahydrate (SrCl₂•6H₂O) (Sigma-Aldrich, catalog number: 255521 )
Sodium nitrate (NaNO₃) (Fisher Scientific, catalog number: S343-500 )
Sodium phosphate monobasic monohydrate (NaH₂PO₄•H₂O) (Sigma-Aldrich, catalog number: S9638 )
Iron(III) chloride hexahydrate (FeCl₃•6H₂O) (Sigma-Aldrich, catalog number: 236489 )
Na₂EDTA•2H₂O (Bio Basic, catalog number: EB0185 )
Copper (II) sulfate pentahydrate (CuSO₄•5H₂O) (Bio Basic, catalog number: CDB0063 )
Sodium molybdate dihydrate (Na₂MoO₄•2H₂O) (Avantor Performance Materials, catalog number: 3764-01 )
Zinc sulfate heptahydrate (ZnSO₄•7H₂O) (Alfa Aesar, catalog number: A12915-36 )
Cobalt (II) chloride hexahydrate (CoCl₂•6H₂O) (Sigma-Aldrich, catalog number: C3169 )
Manganese(II) chloride tetrahydrate (MnCl₂•4H₂O) (BioShop, catalog number: MAN222 )
Selenous acid (H₂SeO₃) (Sigma-Aldrich, catalog number: 229857 )
Nickel(II) sulfate (NiSO₄) (Sigma-Aldrich, catalog number: 656895 )
Sodium orthovanadate (Na₃VO₄) (BioShop, catalog number: SPP310 )
Potassium chromate (K₂CrO₄) (Sigma-Aldrich, catalog number: 216615 )
Thiamine-HCl (Sigma-Aldrich, catalog number: 47858 )
Biotin (Sigma-Aldrich, catalog number: B4639 )
Cyanocobalamin (Sigma-Aldrich, catalog number: C3607 )
Zymogen solution (see Recipes)
Lysis buffer for P. tricornutum cells (see Recipes)
LB medium (see Recipes)
SOC media (see Recipes)
LB agar plates (see Recipes) containing 100 μg ml^-1 ampicillin or 100 μg ml^-1ampicillin with 40 μg ml^-1gentamicin
50% L1, 5% LB, 1% agar plate (1 L) (see Recipes)
L1 media (see Recipes)
L1 agar plates (see Recipes) containing 50 μg ml^-1 Zeocin^TM
2x Aquil salt (see Recipes)
NP stock (see Recipes)
1,000x L1 trace metals (see Recipes)
Vitamin solution (see Recipes)

Equipment

Qubit^® 2.0 Fluorometer (Thermo Fisher Scientific, Invitrogen^TM, model: Qubit^® 2.0 , catalog number: Q32866)
Heat block (VWR, catalog number: 13259-030 )
Water bath (Fisher Scientific, model: IC 2100 )
Micropipettes (PIPETMAN, variable volume)
Binocular Microscope (Leitz Wetzlar, model: Labolux12 )
Hemocytometer
Table top centrifuge (Hettich Lab Technology, catalog number: 2004-01 )
Centrifuge (Beckman Coulter, model: Avanti^® J-26 XPI , catalog number: 393127)
PCR thermal cycler (Bio-Rad Laboratories, model: T100^TM, catalog number: 1861096 )
UV-visible spectrophotometer (Biochrom, model: ULTROSPEC 2100^® , catalog number: 80-2112-21)
Incubator shaker (Thermo Fisher Scientific, model: Large Incubated and Refrigerated, catalog number: SHKE5000-7 )
Vortex
Autoclave
Refrigerator

Procedure

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如何引用

Wang, H., Slattery, S. S., Karas, B. J. and Edgell, D. R. (2018). Delivery of the Cas9 or TevCas9 System into Phaeodactylum tricornutum via Conjugation of Plasmids from a Bacterial Donor. Bio-protocol 8(16): e2974. DOI: 10.21769/BioProtoc.2974.