Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions
通过CRISPR-Cas精确诱导双等位基因缺失以对硅藻进行基因组编辑
发布: 2017年12月05日第7卷第23期 DOI: 10.21769/BioProtoc.2625 浏览次数: 15041
评审: Dennis NürnbergChristian SailerAgnieszka Zienkiewicz
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The authors used this protocol in:
Nov 2016
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Abstract
Genome editing in diatoms has recently been established for the model species Phaeodactylum tricornutum and Thalassiosira pseudonana. The present protocol, although developed for T. pseudonana, can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include how to design a construct using Golden Gate cloning for targeting two sites, allowing a precise deletion to be introduced into the target gene. The transformation protocol is explained, as are the methods for screening using band shift assay and/or restriction site loss.
Keywords: CRISPR-Cas (CRISPR-Cas)
Diatom (硅藻)
Thalassiosira pseudonana (假微型海链藻)
Golden Gate (Golden Gate)
U6 promoter (U6启动子)
Band shift assay (带偏移实验法)
Restriction site loss (限制性位点丢失)
Phaeodactylum tricornutum (三角褐指藻)
Background
CRISPR-Cas is fast becoming a key method for molecular research. Based on a viral defence mechanism found in bacteria and archaea, CRISPR-Cas induces double-strand breaks (DSBs) at precise locations in the genome. It involves the use of a Cas9 nuclease which forms a complex with a chimeric single guide RNA (sgRNA) formed from CRISPR RNA (crRNA) and trans-activating crRNA (trRNA). Specificity is provided by a 20 nt sequence in the crRNA which corresponds with the target in the genome and guides Cas9 to the correct site by base complementarity. This means that the system is easily programmable and can be applied to new targets simply by changing the 20 nt sequence, provided that a protospacer adjacent motif (PAM) is present in the genome directly following the 20 nt target sequence. For the commonly used Cas9 isolated from Streptococcus pyogenes the PAM sequence is NGG. Gene editing is then achieved either by introducing mutations following imperfect repair by non-homologous end joining (NHEJ), cutting two sites and introducing a precise deletion or through homologous recombination. Since its application in the first eukaryotic systems (Cong et al., 2013; Mali et al., 2013), CRISPR-Cas has been used for genome editing in a wide range of organisms including two diatom species (Hopes et al., 2016; Nymark et al., 2016). Nymark et al. (2016) introduced mutations into the genome of Pheodactylum tricornutum using individual sgRNAs–the protocol for which can be found within Bio-protocol (Nymark et al., 2017). The protocol published in this paper focuses on gene editing in Thalassiosira pseudonana using two sgRNAs to introduce a precise deletion and allow simple screening using the band-shift assay previously described for identifying mutants in higher plants (Brooks et al., 2014). In addition, this method uses Golden Gate cloning (Weber et al., 2011; Engler et al., 2014)–a flexible modular system which allows sequences and cassettes to be easily interchanged and multiple modules to be assembled at once. Whilst this protocol describes targeting of two sites in the same gene to introduce a deletion, the construct can easily be altered to target different genes or greater numbers of genes as previously shown by Sakuma et al. (2014), who demonstrated knock-out of 7 genes using the Golden Gate cloning system.
Materials and Reagents
- 0.2 ml PCR tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: AB0620 )
- 10 µl filter pipette tips (STARLAB INTERNATIONAL, catalog number: S1121-3810 )
- 10 µl pipette tips (STARLAB INTERNATIONAL, catalog number: S1111-3810 )
- 200 µl pipette tips (STARLAB INTERNATIONAL, catalog number: S1111-0810 )
- 200 µl filter pipette tips (STARLAB INTERNATIONAL, catalog number: S1120-8810 )
- 1,000 µl pipette tips (STARLAB INTERNATIONAL, catalog number: S1111-6810 )
- 47 mm diameter 1.2 µm isopore filters (Merck, catalog number: RTTP04700 ) (optional)
- 1.5 ml Microcentrifuge tubes (Fisher Scientific, catalog number: 11926955 )
- 90 mm diameter Petri dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 263991 )
- 0.7 µm (M10) tungsten particles (Microcarriers) (Bio-Rad Laboratories, catalog number: 1652266 )
- Culture tubes (Fisher Scientific, catalog number: 11317824 )
- Macrocarriers (Bio-Rad Laboratories, catalog number: 1652335 )
- Macrocarrier holders (Bio-Rad Laboratories, catalog number: 1652322 )
- Stopping screens (Bio-Rad Laboratories, catalog number: 1652336 )
- 1,350 psi rupture discs (Bio-Rad Laboratories, catalog number: 1652330 )
- Thalassiosira pseudonana strain CCMP 1335 (Bigelow) https://ncma.bigelow.org/ccmp1335
- pCRTM8/GW/TOPOTM TA Cloning Kit with One ShotTM TOP10 E. coli (Thermo Fisher Scientific, InvitrogenTM, catalog number: K250020 )
- High efficiency competent E. coli (e.g., NEB 5-alpha Competent E. coli (High Efficiency)) (New England Biolabs, catalog number: C2987H )
- BsaI (New England Biolabs, catalog number: R0535S )
- BpiI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: ER1011 )
- Taq DNA polymerase (e.g., GoTaq Flexi DNA polymerase) (Promega, catalog number: M8291 )
- pICH47732:FCP:NAT (Addgene, catalog number: 85984 ) or pICH47732 (Addgene, catalog number: 48000 )
- pICH47742:FCP:Cas9YFP (Addgene, catalog number: 85986 ) or pICH47742 (Addgene, catalog number: 48001 )
- pICH47751 (Addgene, catalog number: 48002 )
- pICH47761 (Addgene, catalog number: 48003 )
- pICH41780 (Addgene, catalog number: 48019 )
- pAGM4723 (Addgene, catalog number: 48015 )
- pICH86966::AtU6p::sgRNA_PDS (Addgene, catalog number: 46966 )
- pCR8/GW:U6 (Addgene, catalog number: 85981 )
- Q5 Site-Directed Mutagenesis Kit (New England Biolabs, catalog number: E0554S )
- High fidelity DNA polymerase (e.g., Phusion High-Fidelity DNA Polymerase) (New England Biolabs, catalog number: M0530 )
- PCR clean up kit (e.g., Illustra GFX PCR DNA and Gel Band Purification Kit) (GE Healthcare, catalog number: 28-9034-70 )
- Plasmid mini prep kit (e.g., PureYield Plasmid Miniprep System) (Promega, catalog number: A1223 )
- Absolute ethanol (VWR, catalog number: 20821.330 )
- Agarose (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17850 )
- Ethidium bromide for gel electrophoresis (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15585011 )
- TAE buffer for gel electrophoresis (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15558026 )
- T4 DNA ligase (Promega, catalog number: M1794 )
- 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal) (Sigma-Aldrich, catalog number: B4252-50MG )
- IPTG (Sigma-Aldrich, catalog number: I6758-1G )
- Half salinity Aquil media (Price et al., 1989, full recipe at: https://ncma.bigelow.org/algal-recipes)
- Nourseothricin clonNAT (Werner BioAgents)
- Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670-100G )
- Spermidine (Sigma-Aldrich, catalog number: S0266-1G )
- Isopropanol (VWR, catalog number: BDH1133-1LP )
- Compressed Helium supply
- Mix2seq kit (Eurofins)
- Tryptone (Sigma-Aldrich, catalog number: T7293 )
- Yeast extract (ForMedium, catalog number: YEA01 )
- Sodium chloride (Sigma-Aldrich, catalog number: S7653 )
- Agar (ForMedium, catalog number: AGA02 )
- Ampicillin sodium salt (Sigma-Aldrich, catalog number: A0166-5G )
- Spectinomycin dihydrochloride pentahydrate (Sigma-Aldrich, catalog number: S4014-5G )
- Carbenicillin disodium salt (Sigma-Aldrich, catalog number: C3416-250MG )
- Kanamycin sulphate (Sigma-Aldrich, catalog number: 60615-5G )
- Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
- Tris-HCl pH 8.0 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15568025 )
- EDTA (Sigma-Aldrich, catalog number: EDS )
- LB media/LB agar (see Recipes)
- Lysis buffer (see Recipes)
Equipment
- Pipettes (Thermo Fisher Scientific, model: Finnpipette , volumes: 2 µl, 10 µl, 200 µl and 1,000 µl)
- NanoDrop ND-1000 (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 1000 )
- Laminar flow hood (Walker safety cabinets, model: Class II 1290 Recirc Gen 6 )
- Benchtop microfuge (Eppendorf, model: 5418 R )
- Centrifuge (Hettich Lab Technology, model: ROTINA 380 R )
- Autoclave (Prestige Medical, catalog number: 210004 )
- Tweezers
- Vortex (Mo Bio Laboratories, model: Vortex Genie® 2 )
- Shaking incubator (37 °C) (IKA, model: KS 4000 i control )
- Light incubator at 20 °C (Sanyo versatile Environmental Test chamber)
- PCR thermocycler (Bio-Rad Laboratories, model: T100TM Thermal cycler )
- Light Microscope and Neubauer chamber (VWR, catalog number: 631-0696 ) or Coulter Counter (Beckman, model: Multisizer 3 ) for counting cells
- PDS-1000/He biolistic microparticle delivery system (particle gun) (Bio-Rad Laboratories, catalog number: 1652257 )
- Vacuum pump for cell filtration (Welch Vacuum, model: 2534C-02 ) (optional)
- Nalgene filtration funnel (Thermo Fisher Scientific, Thermo ScientificTM, model: DS0320-5045 ) (optional)
- High Vacuum pump for microparticle delivery system (Uniweld Products, model: HUMM•VACTM HVP6 )
- Gel electrophoresis tank (Fisher Scientific, FisherbrandTM, model: Midi Plus Horizontal Gel System )
- Electrophoresis power supply (Consort, model: EV243 )
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Hopes, A., Nekrasov, V., Belshaw, N., Grouneva, I., Kamoun, S. and Mock, T. (2017). Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions. Bio-protocol 7(23): e2625. DOI: 10.21769/BioProtoc.2625.
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分类
微生物学 > 微生物遗传学 > 诱/突变
植物科学 > 藻类学 > DNA > 基因组编辑
分子生物学 > DNA > DNA 修饰
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