PROTOCOLCRISPRmod synthetic guide RNAtransfection protocol for dCas9-VPR ordCas9-SALL1-SDS3 expressing cells
The following is an abbreviated protocol for transfecting CRISPRmod
CRISPRa/CRISPRi synthetic guide RNA into cultured mammalian cells
expressing dCas9-VPR or dCas9-SALL1-SDS3 using DharmaFECT™ 1-4
transfection reagent (Cat. T-2001, T-2002, T-2003, T-2004). Synthetic
guide RNA can be either synthetic single guide RNA, or synthetic
crRNA complexed with tracrRNA. Intended for use after optimization
for your cell line has been completed. For full details, as well as
optimization guidelines please see the appropriate CRISPRmod
technical manual CRISPRa tech manual and CRISPRi technical manual).
This protocol is written for transfection of dCAS9 expressing cells into
96, 24, or 6-well tissue culture plates at 25 nM final concentration ofsynthetic guide RNA.

Tube 1

Tube 2Synthetic
sgRNA,
orcrRNA:tracrRNADiluteDharmafect 1-4transfection reagent
in serum-freemedium
10 µL

10 µL

Incubate at RT for 5 min

96–well protocol
Day 1
Cell plating

Seed cells at a density that is optimal for specific downstream
phenotypic assay(s)Day 2Prepareworking
solutions ofreagents fortransfection

Combineworking
solutions fortransfection
mixtureSynthetic
guide RNA

Dilute sgRNA to a working
concentration of 1 μM in 10 mMTris-HCl, pH 7.4
orDilute and mix crRNA and tracrRNA toa working concentration of 1 μM in
10 mM Tris-HCI, pH 7.4
For one well

For multiple wells

Synthetic guide RNA

2.5 μL_ μL

Serum-free medium

To 10 µL

_ μL

DharmaFECT 1-4
transfection reagent

0.05-0.8 μL__uL

Serum-free medium

To 10 μL_ μL

Tube 1Tube 2Prepare working
solution ofDharmafect 1-4for transfection

Incubate at room temperature for 5 minutes before next step
Combine Tube 1 and Tube 2 and carefully mix by pipeting

Combine Tubes 1 and 2
Carefully mix by pipetting

20 µL

Incubate at RT for 20 min
Add 80 µL antibiotic-freefull growth medium

Incubate at room temperature for 20 minutes before next step
Combinetransfection
mixtureTransfect cells

Add full growthmedium

80 μL

_ μL

Total

100 μL_ μL

Replace growth medium on cells with 100 μL of transfection mixture
Incubate cells for 48-72 hours before performing downstream
phenotypic assay(s) or gene expression analysis.

100 µL

Replace growth medium on
cells with transfection mixture

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24–well protocol

6–well protocol

Day 1

Day 1

Cell plating

Seed cells at a density that is optimal for specific downstream
phenotypic assay(s)Combineworking
solutions fortransfection
mixtureSynthetic
guide RNA

Dilute sgRNA to a working
concentration of 1 μM in
10 mM Tris-HCl, pH 7.4
orDilute and mix crRNA and tracrRNA toa working concentration of 1 μM in10 mM Tris-HCl, pH7.4
For one well

For multiple wells

Tube 1Synthetic guide RNA

12.5 uL_ μL

Serum-free medium

To 50 µL

_ μL

Prepareworking
solutions ofreagents fortransfection

Combineworking
solutions fortransfection
mixtureDharmaFECT 1-4

0.24 - 4 uL

_ μL

Serum-free medium

To 50 μL

_ μL

Incubate at room temperature for 5 minutes before next step

Prepareworking
solution of
Dharmafect 1-4for transfection

For multiple wells

Synthetic guide RNA50 uL_ μL

Serum-free medium

To 200 uL_ μL

DharmaFECT 1-4

1-20 uL_ μL

Serum-free medium

To 250 μL_ μL

Add full growth
medium

400 μL

_ μL

Total

500 μL_ μL

Replace growth medium on cells with 500 uL of transfection mixture

Incubate at room temperature for 5 minutes before next step

Incubate at room temperature for 20 minutes before next step

Incubate at room temperature for 20 minutes before next step

Transfect cells

For one well
Tube 1

Combine Tube 1 and Tube 2 and carefully mix by pipeting

Combine Tube 1 and Tube 2 and carefully mix by pipeting

Combinetransfection
mixtureSynthetic
guide RNA

Dilute sgRNA to a working
concentration of 1 μM in
10 mM Tris-HCl, pH 7.4
orDilute and mix crRNA and tracrRNA toa working concentration of 1 μM in
10 mM Tris-HCl, pH7.4

Tube 2

Tube 2
Prepareworking
solution ofDharmafect 1-4
for transfection

Seed cells at a density that is optimal for specific downstream
phenotypic assay(s)

Day 2

Day 2
Prepareworking
solutions ofreagents fortransfection

Cell plating

Combinetransfection
mixtureTransfect cells

Incubate cells for 48-72 hours before performing downstream phenotypic assay(s)or gene expression analysis.

Add full growthmedium

1600 uL_ μL

Total

2000 uL_ μL

Replace growth medium on cells with 2000 uL of transfection mixture
Incubate cells for 48-72 hours before performing downstream phenotypic assay(s)or gene expression analysis.

For more information

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