The Transfection &Gene Expression Experts

Avalanche®-CRISPR Transfection Reagent
Cat. No. EZT-CRPR-1

Size: 0.5 ml
1.5 mlStore at 4°C

CRISPR-Cas9–mediated genome targeting:
CRISPR-Cas9 has triggered a revolution in which laboratories around the world are using thetechnology for innovative applications in biology. This technology is able to systematicallyanalyze gene functions in mammalian cells, study genomic rearrangements and theprogression of cancers or other diseases, and potentially correct genetic mutations
responsible for inherited disorders.
The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign
genetic elements such as plasmids and phages, and provides a form of acquired immunity.
CRISPR spacers recognize and cut these exogenous genetic elements in a manner analogous
to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% ofsequenced bacteria genomes and 90% of sequenced archaea.
The CRISPR interference technique can be applied in eukaryotic cells. By delivering the Cas9 protein
and appropriate guide RNAs (sgRNA) into a cell, the organism's genome can be cut or modified at any
desired locations.

The Transfection Reagent:
One of the most important factors that determines a successful CRISPR-Cas9–mediated genome
editing is the efficiency of delivering functional Cas9 gene and sgRNA into the cells (the transfectionprocess). EZ Biosystems, the transfection and gene expression experts, has specifically designed, andsuccessfully developed a transfection reagent for CRISPR-Cas9 gene editing in mammalian cells ---Avalanche®-CRISPR Transfection Reagent.

Features:Specifically designed for CRISPR-Cas9 gene editing transfection in mammalian cells
Maximum transfection efficiency for transfecting large plasmids (>9.0 kb)
Great for co-transfection of large plasmids with either small plasmids or small RNA, such assgRNAAvalanche®-CRISPR Transfection ReagentGreat for CRISPR-Cas9 gene editing in difficult-to-transfect cells, such as primary cells,suspension culture cells, or neural cells.
Lowest Cellular Toxicity-maintain cell density and reduce experimental biases
Compatible with serum
Chemically defined compounds and completely free of animal‐derived components.
2BEFORE YOU START:Important Tips for Optimal TransfectionPrepare high-quality pCas9 expression plasmids and sgRNA expression plasmids at
1.0–5 μg/μl in deionized water or TE buffer.

Use Opti-MEM® I Reduced Serum Medium (Life Technologies) or regular DMEM without
serum to make The Reagent and nucleic acid mix. Do not use NaCl2 solution or PBS.

Maintain the same seeding conditions between experiments. Use low-passage cells;
make sure that cells are healthy and greater than 90% viable before transfection.
The Reagent is extremely gentle to cells. However, transfection process will impose
stress on cells, no matter what type of transfection methods you use. The trick is to get
the balance between transfection efficiency and cell viability. Increasing the number ofcells plated per well or decreasing The Reagent amount will minimize the effect oftransfection on cell growth and viability. With careful adjustment, as described in page 3and 4, this can be achieved while keeping the highest transfection efficiency.
Don’t use antibiotics in the culture medium during the first 24 hours of transfection.

EZ Biosystems LLC ∙ 8400 Baltimore Ave ∙ College Park ∙ MD ∙ 20740 ∙ Phone: 800 614 6133 ∙ FAX: 240 241 7046

www.ezbiosystems.com

Avalanche®-CRISPR Transfection Reagent

Protocols
1. Cell Seeding
For optimal DNA transfection conditions, we recommend using cells which are 70% to 90%
confluent at the time of transfection. Typically, for experiments in 6-well plates, 150,000250,000 adherent cells are seeded per well in 2 ml of cell growth medium without
antibiotics 24 h prior to transfection. For the different culture formats, refer to Table 1.
Table 1. Recommended number of cells to seed the day before transfection in culture medium
without antibiotics
Number ofVolume of medium
Surface area
Culture vessel
Adherent cells to seed
per well to seed
per well (cm2)(Suspension Cells)
the cells (ml)50,000-80,000
24-well
1.90.5(2x105)
80,000-150,000
12-well
3.81(4x105)
150,000-250,000
6-well/35 mm
9.42(8x105)
250,000-800,000
60 mm/flask 25 cm225-285(2x106)
1x106-2x106
100 mm/flask 75 cm275-78.510(6x106)
2x106-5x106
150 mm/flask 175 cm2
153-17525(1.3x107)
2. Transfection
 Change with fresh media for the cells before transfection.
 Mix pCas9 expression plasmid and sgRNA expression plasmids at the ratios of 1:1 ̴
1:4 (w/w) or 1:2 ̴ 1:10 (molar ratio). The optimal ratios vary based on the propertiesof different Cas9 expression plasmids and sgRNA expression plasmids, and should be
empirically determined.
 If this is the first time that you are using Avalanche®-CRISPR Transfection Reagent ona specific type of cells, first, transfect the cells according to Table 2a for optimization(The optimization procedures are extremely important for successful transfection.
Since different types of cells have different sensitivity to Avalanche®-CRISPR
Transfection Reagent, the amount of Avalanche®-CRISPR Transfection Reagent
needed for maximum transfection on different types of cells may differdramatically).
EZ Biosystems LLC ∙ 8400 Baltimore Ave ∙ College Park ∙ MD ∙ 20740 ∙ Phone: 800 614 6133 ∙ FAX: 240 241 7046

www.ezbiosystems.com

3

Avalanche®-CRISPR Transfection Reagent
Table 2a. Transfection optimization guidelines according to the cell culture vessel per well
Component

24-well

6-well

Opti-MEM Medium (µl)

250

1200

Total plasmid DNA (µg)

2.512Diluted plasmid DNA (µl)

4 x 50 µl

4 x 250 µl*0.2, *0.4, *0.7, 1.0

1.0, 2.0, 3.5, 5.0

Avalanche®-CRISPR Transfection
Reagent (µl)

Incubate for 15 minutes at room temperature
DNA-reagent complex/well (µl)

50

250

Immediately centrifuge the plate at 300 g for 5 min
Gently put in incubator, and Incubate cells for 48 hours or more at 37°C

*Dilute Avalanche®-CRISPR Transfection Reagent 1:5 with H2O prior to application (4 µl reagent + 16µl H2O), and then use 5 times of the volumes in the table for accurate pipetting.

Table 2b shows the amounts of total DNA and Avalanche®-CRISPR per well used in each of
the above transfection reactions.
Table 2b. Amount of total DNA and Avalanche®-CRISPR per wellAmount24-well

6-wellTotal DNA/well (ng)

500

2500

Avalanche®-CRISPR (µl)

0.2-1.0

1.0-5.0

As an example, the following steps are given for optimization on 6-well plate. For otherculture formats, please refer to Table 2 and Table 3.
1) Transfer 12.0 μg total DNA into 1200 μl Opti-MEM® Reduced-Serum Medium (Cat#31985-070, Life Technologies) or regular high glucose DMEM without serum. Mix byvortexing. Aliquot 4 x 250 μl of the above DNA solution into 4 x 1.5 ml Eppendorftubes.
2) Briefly vortex Avalanche®-CRISPR Transfection Reagent, and add 1.0, 2.0, 3.5, and 5.0
μl into the above diluted DNA respectively. Immediately vortex for 5 s after eachaddition.
3) Incubate for 15 min at RT.
4) Add the 250 µl transfection mixture drop-wise into each well.
5) Gently rock the plates back and forth and from side to side, and immediately
centrifuge the plate at 300 g for 5 min.
EZ Biosystems LLC ∙ 8400 Baltimore Ave ∙ College Park ∙ MD ∙ 20740 ∙ Phone: 800 614 6133 ∙ FAX: 240 241 7046

www.ezbiosystems.com

4Avalanche®-CRISPR Transfection Reagent
6) Gently put in incubator, and incubate at 37 °C CO2. It is not necessary to remove
complexes or to change/add medium after transfection.
7) Cells were incubated at 37ºC for 48 hours or more post transfection before genomic
DNA extraction for analysis or cell selection/isolation
After you have completed the optimization steps, choose the amount of Avalanche®-CRISPRTransfection Reagent that gave you the optimal balance of potency & low cytotoxicity for allof your future experiments on this specific cell type of cells.
3. Scale Up or Down Transfections
Use Table 3 to scale the volumes for your transfection experiment.
Table 3. Scaling Up Transfection Instruction
Multiplication
Vol. Complex-Opti- Total DNA Avalanche®-CRISPR
Culture Vessel(µl)
factor1
MEM per well (µl)(µg)
24-well
1.00
50
0.5
0.2-1.0
12-well
2.00
100
1.0
0.4-2.0
6-well5.00
200
2.5
1.0-5.0
60-mm
11.05
500
5.5
2.3-11.5
10-cm28.95
1000
145.8-29
T75
39.47
1500
207.9-39
1After determining the optimum reagent amount, use the Multiplication factor todetermine the reagent amount needed for your new plate format.
2Optimum amount needed is determined from the protocol in the previous two pages.

EZ Biosystems LLC ∙ 8400 Baltimore Ave ∙ College Park ∙ MD ∙ 20740 ∙ Phone: 800 614 6133 ∙ FAX: 240 241 7046

www.ezbiosystems.com

5

Avalanche®-CRISPR Transfection Reagent

Intended Use:All Avalanche® Series Transfection Reagents are for research use only, not intended for any animal orhuman therapeutic or diagnostic use.

Avalanche® is the trademark of EZ Biosystems LLC. All rights reserved.
Avalanche® series Transfection Reagents are sold to the Buyer with a limited license for Research Use
Only and is not for clinical, therapeutic or diagnostic use in humans or animals.
Avalanche® series Transfection Reagents may not be re-packaged or re-sold without written
permission from EZ Biosystems LLC. The buyer agrees not to attempt to reverse engineer,reconstruct, synthesize or otherwise modify EZ Biosystems Avalanche® products. A license from EZBiosystems LLC is required for commercial application of Avalanche® series Transfection Reagents.
For obtaining a license to use those products for commercial application, contact EZ Biosystems LLC
via business@ezbiosystems.com. For full terms and conditions, visit: www.ezbiosystems.com.

EZ Biosystems LLC ∙ 8400 Baltimore Ave ∙ College Park ∙ MD ∙ 20740 ∙ Phone: 800 614 6133 ∙ FAX: 240 241 7046

www.ezbiosystems.com

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