參考文獻(xiàn)：

Genome engineering using the CRISPR-Cas9 system

Nature protocols, 2013  Nov; 8(11): 2281–2308.

F Ann Ran, Patrick D Hsu, Jason Wright, Vineeta Agarwala, David A Scott,and Feng Zhang

被引用次數(shù)：2103

CRISPR-Cas9 基因編輯流程

試劑

sgRNA制備

Plasmids: pSpCas9 (Addgene plasmid ID: 48137), pSpCas9(BB) (formerly pX330; Addgene plasmid ID: 42230), pSpCas9(BB)-2A-GFP (Addgene plasmid ID: 48138), pSpCas9(BB)-2A-Puro (Addgene plasmid ID: 48139), pSpCas9n(BB) (Addgene plasmid ID: 48873), pSpCas9n(BB)-2A-GFP (Addgene plasmid ID: 48140), pSpCas9n(BB)-2A-Puro (Addgene plasmid ID: 48141).

pUC19 (Invitrogen, cat. no. 15364-011) or any preferred cloning plasmid

PCR primers or oligos for sgRNA construction are listed in Table 1 and in supplementary Data 1. Primers longer than 60 bp can be ordered as 4-nmol ultramers (Integrated DNA Technologies)

UltraPure DNase/RNase-free distilled water (Life Technologies, cat. no. 10977-023)

Herculase II fusion polymerase with 5× reaction buffer (Agilent Technologies, cat. no. 600679) ▲ CRITICAL To minimize error in amplifying sgRNAs, it is important to use a high-fidelity polymerase. Other high-fidelity polymerases, such as PfuUltra (Agilent) or Kapa HiFi (Kapa Biosystems), may be used as substitutes.

Taq DNA polymerase with standard Taq buffer (NEB, cat. no. M0273S)

dNTP solution mix, 25 mM each (Enzymatics, cat. no. N205L)

MgCl2, 25 mM (Thermo Scientific, cat. no. R0971)

QIAquick gel extraction kit (Qiagen, cat. no. 28704)

QIAprep spin miniprep kit (Qiagen, cat. no. 27106)

UltraPure TBE buffer, 10× (Life Technologies, cat. no. 15581-028)

SeaKem LE agarose (Lonza, cat. no. 50004)

SYBR Safe DNA stain, 10,000× (Life Technologies, cat. no. S33102)

1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018)

TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028)

FastDigest BbsI (BpiI) (Fermentas/Thermo Scientific, cat. no. FD1014)

Fermentas Tango buffer (Fermentas/Thermo Scientific, cat. no. BY5)

DTT (Fermentas/Thermo Scientific, cat. no. R0862)

T7 DNA ligase with 2× rapid ligation buffer (Enzymatics, cat. no. L602L). Alternative ligases, such as T4 DNA ligase, can also be used. If you are using other ligases, substitute with the compatible buffer

T4 polynucleotide kinase (New England BioLabs, cat. no. M0201S)

T4 DNA ligase reaction buffer, 10× (New England BioLabs, cat. no. B0202S)

Adenosine 5′-triphosphate, 10 mM (New England BioLabs, cat. no. P0756S)

PlasmidSafe ATP-dependent DNase 

One Shot Stbl3 chemically competent E. coli (Life Technologies, cat. no. C7373-03)

SOC medium (New England BioLabs, cat. no. B9020S)

LB medium (Sigma, cat. no. L3022)

LB agar medium (Sigma, cat. no. L2897)

Ampicillin, 100 mg ml–1, sterile filtered (Sigma, cat. no. A5354)

哺乳動(dòng)物細(xì)胞培養(yǎng)

細(xì)胞HEK 293FT cells (Life Technologies, cat. no. R700-07)

細(xì)胞系HUES 9 cell line (Harvard Stem Cell Science)

高糖培養(yǎng)基DMEM, high glucose (Life Technologies, cat. no. 10313-039)

高糖培養(yǎng)基，無(wú)酚紅DMEM, high glucose, no phenol red (Life Technologies, cat. no. 31053-028)

磷酸緩沖液Dulbecco's PBS (DPBS; Life Technologies, cat. no. 14190-250)

胎牛血清FBS, qualified and heat inactivated (Life Technologies, cat. no. 10438-034)

Opti-MEM I reduced-serum medium (Life Technologies, cat. no. 11058-021)

Penicillin-streptomycin, 100× (Life Technologies, cat. no. 15140-163)

Puromycin dihydrochloride (Life Technologies, cat. no. A11138-03)

TrypLE Express, no phenol red (Life Technologies, cat. no. 12604-013)

Lipofectamine 2000 transfection reagent (Life Technologies, cat. no. 11668027)

Amaxa SF cell line 4D-Nucleofector X kit S, 32 RCT (Lonza, cat. no. V4XC-2032)

Geltrex LDEV-free reduced growth factor basement membrane matrix (Life Technologies, cat. no. A1413201)

培養(yǎng)基mTeSR1 medium (Stemcell Technologies, cat. no. 05850)

Normocin (InvivoGen, cat. no. ant-nr-1)

Accutase cell detachment solution (Stemcell Technologies, cat. no. 07920)

Rho-associated protein kinase (ROCK) inhibitor (Y-27632; Millipore, cat. no. SCM075)

Amaxa P3 primary cell 4D-Nucleofector X kit S, 32 RCT (Lonza, cat. no. V4XP-3032)

Genotyping analysis基因分型分析

PCR引物PCR primers for SURVEYOR, RFLP analysis or sequencing; see Table 1, supplementary Data 1 (alternatively, they can be custom made)

QuickExtract DNA extraction solution 

SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025)

TBE Gels, 4–20%, 1.0 mm, 15 well (Life Technologies, cat. no. C62255BOX)

Novex Hi-Density TBE sample buffer, 5× (Life Technologies, cat. no. LC6678)

SYBR Gold nucleic acid gel stain, 10,000× (Life Technologies, cat. no. S-11494)

FastDigest HindIII (Fermentas/Thermo Scientific, cat. no. FD0504)

FastDigest buffer, 10× (Fermentas/Thermo Scientific, supplied with FastDigest HindIII)

FastAP Antarctic phosphatase (Fermentas/Thermo Scientific, cat. no. EF0654)

Nextera XT index kit (Illumina, cat. no. FC-131-1001)

儀器耗材

Filtered sterile pipette tips (Corning)

標(biāo)準(zhǔn)離心管1.5 ml

PCR板Axygen PCR plates, 96 well (VWR, cat. no. PCR-96M2-HSC)

Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250)

Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097)

Falcon tubes, polypropylene, 50 ml (BD Falcon, cat. no. 352070)

Round-bottom tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235)

Petri dishes, 60 mm × 15 mm (BD Biosciences, cat. no. 351007)

Tissue culture plate, 24 wells (BD Falcon, cat. no. 353047)

Tissue culture plate, 96 wells flat bottom (BD Falcon, cat. no. 353075)

Tissue culture dish, 100 mm (BD Falcon, cat. no. 353003)

Nunc EasYFlask 225 cm2 (T225 flask), filter cap, 70-ml working volume (Thermo Scientific, cat. no. 159934)

Nunc EasYFlask 75 cm2 (T75 flask), filter cap, 25-ml working volume (Thermo Scientific, cat. no. 156499)

INCYTO C-Chip disposable hemocytometer (VWR, cat. no. 82030-468)

Steriflip-GP Filter Unit, 0.22 μM (Millipore, cat. no. SCGP00525)

Thermocycler with programmable temperature stepping functionality, 96 well (Applied Biosystems Veriti, cat. no. 4375786)

桌面離心機(jī)

Gel electrophoresis system (PowerPac basic power supply, Bio-Rad, cat. no. 164-5050), and Sub-Cell GT System gel tray (Bio-Rad, cat. no. 170-4401)

Novex XCell SureLock mini-cell (Life Technologies, cat. no. EI0001)

Digital gel imaging system (GelDoc EZ, Bio-Rad, cat. no. 170-8270), and blue sample tray (Bio-Rad, cat. no. 170-8273)

Blue-light transilluminator and orange filter goggles (SafeImager 2.0; Invitrogen, cat. no. G6600)

Gel quantification software (Bio-Rad, ImageLab or open-source ImageJ from the National Institutes of Health (NIH), USA, available at http://rsbweb.nih.gov/ij/)

UV spectrophotometer (NanoDrop 2000c, Thermo Scientific)

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