GKO v2 User Manual

USER MANUAL
Gene Knockout Kit v2
Gene Knockout Kit v2 Synthego.com/contact

For Research Purposes Only. © 2019 (888) 611-6883
1
USER MANUAL
Table of Contents
Product Overview 3
Representative Data 4
Materials Provided 4
Additional Materials Required 4
Workflow Schematic 5
Transfection Protocols 6
Nucleofection 7 - 10
Lipofection 10 - 13
Troubleshooting Guide 14
Nucleofection 14
Lipofection 15
Read complete user manual before using the kit.

2
USER MANUAL
Product Overview
CRISPR-Cas9 is a precision genome editing tool used by scientists to create a wide array of genetic modification in
a variety of organisms and cell types. This system consists of a Cas9 nuclease and a single guide RNA (sgRNA) that
directs the nuclease to cut at a precise location on the cell’s genome (Fig 1). These double stranded breaks (DSBs) in
the DNA enable genomic edits to be made, including gene knockouts, knock-ins, and single base-pair changes.
Cas9
Figure 1. The CRISPR-Cas9 system.
The CRISPR-Cas9 system is composed of a single guide RNA
(sgRNA) and Cas9, which together form a ribonucleoprotein (RNP)
sgRNA
complex. The sgRNA sequence binds to the complementary
sequence of the genomic target upstream of a protospacer
5' 3' adjacent motif (PAM). The Cas9 nuclease then makes a double-
N
CC
3' 5'
G
stranded break (DSB) in the DNA (denoted by the scissors).

G
N
Target DNA PAM
Synthego’s Gene Knockout Kit v2 is specifically designed to generate a knockout of a single human protein-coding
gene, guaranteed!+ We utilize a multi-guide strategy, in which up to three sgRNAs are strategically designed to
target your gene of interest.* The multiple sgRNAs induce multiple concurrent DSBs in the target sequence, which
consequently cause one or more 21+ bp deletions (Fig 2). These highly disruptive “fragment deletions” reliably
knock out the target gene.
Wild Type
Multi-guide
Figure 2. Synthego's multi-guide design.

Synthego’s multi-guide sgRNA design includes up to 3 modified sgRNAs (grey bars) that target a single gene of interest. Each sgRNA binds
to a complementary sequence that is upstream of a PAM sequence particular to SpCas9 (5’-NGG-3’ or 5’-NGA-3’; underlined in black).
When co-transfected, the sgRNAs create concurrent double-stranded breaks (vertical dotted lines) at the targeted genomic locus and
consequently induce one or more 21+ bp fragment deletions. These large deletions (~7 amino acids) effectively terminate protein function.
The Gene Knockout Kit v2 is effective in a variety of cell types, including primary and stem cells. All sgRNAs are
designed to be used with SpCas9 nuclease (S. pyogenes) and contain chemical modifications that resist intracellular
degradation and prevent activation of intracellular immune responses. This user manual contains two protocols
(nucleofection and lipofection) for transfection of multi-guide sgRNA as ribonucleoprotein (RNP) complexes.
+ For details on Synthego’s money-back guarantee, please visit Synthego.com/legal/money-back-guarantees.

* For some genetic targets, using one sgRNA may be identified as the optimal strategy for achieving a knockout. In these cases, only one
sgRNA will be included in the Gene Knockout Kit v2.

3
USER MANUAL
Representative Data
ALPK3 JAK1 NUAK2
100
Figure 3. Multi-guide sgRNA achieves high
knockout efficiencies.
Three single guide RNAs were designed for each
80
of three genes (ALPK3, JAK1, NUAK2) and introduced
individually (sgRNA1, sgRNA2, sgRNA3) and together
(multi-guide). On average, the multi-guide sgRNA
KO Score (%)
60
performed 50.8%, 32.1%, and 48.2% better than
individual guides for ALPK3, JAK1, and NUAK2,
40 respectively. HEK293 cells (for ALPK3) and MCF7
cells (for JAK1 and NUAK2) were transfected with
ribonucleoproteins (RNPs) via nucleofection. The
20 region around each target was PCR-amplified,
Sanger-sequenced, and analyzed using Inference
of CRISPR Edits (ICE) analysis. Knockout (KO) Score
0
refers to the percentage of sequences that result in
A1
A2
ti- 3
A1
A2
ti- 3
A1
A2
ti- 3
e
a putative knockout (frameshift-inducing indels and
id
id
id
A
A
RN
RN
RN
RN
RN
RN
RN
RN
RN
gu
gu
gu
sg
sg
sg
sg
sg
sg
sg
sg
sg
21+ bp fragment deletions).

ul
ul
ul
M
Materials Provided
Quantity Name Description Storage
1.5 nmol Target specific multi-guide sgRNA 1 - 3 chemically modified sgRNA(s) (in 1 tube) -20°C for up to 3 months
(6 months if not repeatedly
thawed).
1.5 ml Nuclease-free Tris-EDTA Buffer 10 mM Tris, 1 mM EDTA (pH 8.0) Room temperature
(1X TE buffer)
1.5 ml Nuclease-free water - Room temperature
Additional Materials Required

Name Description Ordering Information
Cas9 2NLS nuclease Wild type Cas9 from S. pyogenes Synthego (available at checkout)
(20 µM, 162 kDA)
Transfection Optimization Kit Includes: Synthego (available at checkout)

(recommended) Positive control multi-guide sgRNA
(human TRAC)
Positive control PCR and sequencing primers
(human TRAC)
Nuclease-free Tris-EDTA Buffer
Nuclease-free water
Cas9 NLS nuclease from S. pyogenes
(300 pmol, 20 µM, 162 kDA)
Look through the entire user manual for a complete list of materials needed for conducting and analyzing
a complete experiment. Additional materials are listed in each of the transfection protocols (nucleofection &
lipofection).

4
USER MANUAL
Workflow Schematic
Optimization of Transfection
Transfection Using Positive Control Multi-guide sgRNA
4 DAYS PCR & Sanger Sequencing
ICE Analysis
Target Knockout Experiment
Transfection Using Target Multi-guide sgRNA
4 DAYS PCR & Sanger Sequencing
ICE Analysis
Clonal Isolation of Knockout

(optional)
2 - 8 W E E KS Limiting Dilution
Clonal Expansion
Note: This user manual contains two transfection protocols (nucleofection and lipofection). For instructions on PCR, Sanger
sequencing, and ICE analysis, see Synthego's Knockout Analysis protocol. For instuctions on clonal isolation, see Synthego's
Limiting Dilution and Clonal Expansion protocol.

5
USER MANUAL
Transfection Protocols
All protocols outlined have been validated using materials mentioned in this manual. Materials other than the
ones outlined in our manual may require additional optimization by the user.
General Guidelines
• Wearing gloves and using nuclease-free tubes and reagents are recommended in order to avoid RNase
contamination.
• Always maintain sterile technique, and use sterile, filter pipette tips.
• The GFP transfection efficiency (Transfection control in the table below) should be assessed using
fluorescence microscopy 48-72 hours post-transfection. This condition can be discarded after establishing the
percentage of GFP positive cells. This optimization should be done once for each cell type.
• Synthego highly recommends optimizing transfection conditions for your cell type using TRAC multi-guide
sgRNA prior to conducting your knockout experiment
(available in Synthego’s Transfection Optimization Kit, pg 4).
• For instructions on how to analyze editing efficiency, see Synthego’s Knockout Analysis protocol.
• For instructions on how to clone knockout cells, see Synthego’s Limiting Dilution and Clonal Expansion
protocol.
Suggested Controls
Note: Positive and Transfection controls must be ordered separately.
Control Description Purpose
Mock No Cas9 or sgRNA Wild type sequence for comparison with experimental and
other negative controls. Controls for toxicity from RNP, cell
death from transfection, or possible viability
Negative control Cas9 complexed with a non-targeting Ensures that there are no false positives due to
sgRNA or no sgRNA contamination (no effect expected=wild type). Issues
associated with editing the specific gene of interest.
Positive control sgRNA that has validated high editing Ensures that all reagents, protocol, and equipment are
efficiency. functioning (effect expected).
Recommended:
human TRAC multi-guide sgRNA: Used to optimize transfection conditions for a particular
sgRNA 1: cell type.
5'-CUCUCAGCUGGUACACGGCA-3'
sgRNA 2:
5'-GAGAAUCAAAAUCGGUGAAU-3'
sgRNA 3:
5'-ACAAAACUGUGCUAGACAUG-3'
Transfection pMAXGFPTM vector Assess transfection efficiency (without the use of RNPs).

6
USER MANUAL
Nucleofection Protocol
This protocol is meant to provide a starting point for your CRISPR editing experiments and has been optimized for
Nucleofection of 150,000 HEK293 cells using a 9:1 sgRNA to Cas9 ratio. For specific nucleofection settings for your
cell type, we suggest consulting the Lonza Nucleofector™ cell and transfection database, available online at:
knowledge.lonza.com. All Synthego and Nucleofector™ reagents should be stored according to the manufacturer’s
recommendations. We highly recommend optimizing transfection conditions to your cell type using a positive
control multi-guide sgRNA (e.g., Transfection Optimization Kit) prior to performing your knockout experiment.
Optimization of editing efficiency for a specific cell type will require varying the following:
• The number of cells per reaction
• Amount of Cas9
• Ratio of sgRNA:Cas9
• Nucleofection program
• Type of Nucleofection Solution
Material Purpose
Positive control multi-guide sgRNA (optional) Recommended:Transfection Optimization Kit

(Synthego, available at checkout)
Transfection control (optional) Recommended: pMAXGFPTM (Lonza)
Normal growth medium Cell-type dependent
24-well cell culture plates Corning, Catalog #3526
TrypLE Express or preferred cell dissociation reagent Multiple vendors (e.g., Thermo Fisher Scientific)
1X PBS, cell culture grade Multiple vendors (e.g., Thermo Fisher Scientific, Lonza)
Cell counter Multiple vendors (e.g., Thermo Fisher Scientific)
Microcentrifuge tubes Multiple vendors (e.g., Eppendorf)
4D-Nucleofector™ System (4D-Nucleofector™ Core Unit and Lonza

4D-Nucleofector™ X Unit)
Cell specific Lonza 4D-Nucleofector™ X Kit with 16-well Lonza

Nucleocuvette™ Strips

7
USER MANUAL
1. Pre-Nucleofection
1.1 Seed Cells
a. Subculture cells 2-3 days before nucleofection and seed them in an appropriately sized vessel so that they are
70-80% confluent on the day of transfection. Each nucleofection reaction will require 150,000 cells.
Note: As a general rule, it is recommended to use cells at the lowest passage number possible.
2. Setup & Nucleofection

2.1 Prepare Destination Plates
a. Pre-warm 1 ml of normal growth medium in each well of two 24-well cell culture plates. After nucleofection, the
cells will be split into two wells on the duplicate plates. The cells on the first plate will be lysed and processed to
analyze editing efficiency. The cells on the second plate will be cultured for use in assays, banking, and/or single-
cell cloning. Consider whether you plan to culture control cells on the second plate.
2.2 Dissolve and Dilute your RNA
a. Briefly centrifuge your tubes containing the 1.5 nmol multi-guide sgRNA to ensure that the dried RNA pellet is
collected at the bottom.
b. Rehydrate sgRNA in 15 µl nuclease-free buffer (1X TE buffer) and pulse vortex for 30 seconds to ensure
complete mixing. This will make a stock solution of 100 µM (100 pmol/µl) of multi-guide sgRNA.
Note: If not being used immediately, dissolved multi-guide sgRNA should be aliquoted into 6 µl per tube and stored at
-20 °C. Under these conditions, the provided sgRNA is stable for 3 months (6 months if not thawed repeatedly).
c. Add 6 µl of 100 µM multi-guide sgRNA to 14 µl of nuclease-free water to make a total volume of 20 µl of 30 µM
multi-guide sgRNA (30 pmol/µl). Pulse vortex for 30 seconds and incubate at room temperature for 5 minutes to
dissolve the sgRNA.
2.3 Assemble Ribonucleoprotein (RNP) Complexes (9:1 sgRNA to Cas9 ratio)
a. Ensure Cas9 2NLS is at a concentration of 20 µM (20 pmol/µl; 3.22 mg/ml).
Note: Cas9 2NLS from Synthego is at a concentration of 20 µM and does not require further dilution.
b. Make sure that the entire supplement is added to the Nucleofector™ Solution. The ratio of Nucleofector™
Solution to supplement is 4.5:1.
c. Label and add the reagents to a 0.2 ml PCR tube-strip or similar, in the order shown in the table on page
9. The sgRNA: Cas9 ratio in the table is 9:1, but you may need to experimentally determine the optimum
sgRNA:Cas9 ratio for your cell type or experiment. Synthego recommends sgRNA:Cas9 ratios between 3:1
and 9:1. The RNPs should be formed directly in Nucleofector™ Solution (at room temperature).
Controls Experimental
Reagents
Negative Positive
Transfection (GFP) Mock Target multi-guide sgRNA
(Cas9 only) (TRAC multi-guide sgRNA)
Nucleofector™ Solution 24.6 µl 25 µl 24 µl 18 µl 18 µl

+ Supplement
pmaxGFP vector 0.4 µl - - - -

(1 µg/µl)
Multi-guide sgRNA - - - 6 µl 6 µl
(30 pmol/µl)
Cas9 2NLS nuclease - - 1 µl 1 µl 1 µl

(20 pmol/µl)
Total volume 25 µl 25 µl 25 µl 25 µl 25 µl

8
USER MANUAL
d. Incubate RNPs for 10 minutes at room temperature. Keep at room temperature for up to 1 hour for use, store
at 4°C for up to one week, or at -20°C for up to 1 month. If immediately transfecting cells, a 5 µl cell suspension
(prepared in 2.4 below) will be added to the 25 µl of pre-complexed RNPs for a total transfection volume of 30 µl
per reaction.
2.4 Prepare Cells
Note: For suspension cells, spin down cells before aspiration of culture medium and washes (step a below). Skip
steps b and c below. Resuspend in growth medium for counting.
a. Aspirate cell culture media and wash cells 1-2 times with appropriate volume of 1X PBS.
b. Add appropriate amount of TrypLE Express, or preferred dissociation reagent, and incubate the cells at 37°C/5%
CO2 for 5 minutes, or until they detach from the plate completely. Do not shake or hit the flask to dislodge cells,
as this may lead to clumping and inaccuracies in cell counting and inefficient transfection.
c. Neutralize the dissociation reaction with at least 2 volumes of normal growth medium.
d. Count the cells to determine the cell density.
e. Prepare a cell suspension in Nucleofector™ Solution to 30,000 cells per µl. Each reaction will require 150,000
cells.
For example: For a cell suspension for 18 transfections (16 reactions + 2 extra), each with 150,000 cells, centrifuge
2,700,000 cells at 90 x g for 10 minutes, aspirate media and resuspend the cell pellet carefully in 90 µl of Nucleofector™
Solution (30,000 cells/µl).
2.5 Prepare Cell/RNP Solution

a. For each reaction, add 5 µl of cell suspension to 25 µl of pre-complexed RNP for a total transfection volume of
30 µl.
b. Transfer all 30 µl of cell-RNP solution to Nucleocuvette™ strips and click the lid into place.
c. Gently tap the Nucleocuvette™ Vessels on the benchtop to make sure the sample covers the bottom of the
cuvette and that there are no bubbles in the cuvette.
Note: While pipetting, the cell suspension needs frequent/gentle agitation to prevent the cells from settling. Work
quickly, but carefully, and avoid leaving cells in Nucleofector™ Solution for longer than 15 minutes. Avoid bubble
formation.
2.6 Transfect Cells
a. Program the Nucleofector with appropriate program code (CM-130 for HEK293).
b. Place the Nucleocuvette™ Vessel with closed lid into the retainer of the 4D-X Core unit. Check for proper
orientation of the Nucleocuvette™ Vessel. Larger cutout is the top (A1 and A2) and the smaller cutout is the
bottom (H1 and H2).
c. Press “Start” on the display of the core unit.
d. After run completion, the screen should display a green “+” over the wells that were successfully transfected.
Remove the cuvette strips from the Core unit.
Note: Some cell types require a 10-minute incubation at room temperature after nucleofection. Please consult the
optimized Lonza protocol to see if this is a necessary step for your cell line.
2.7 Add Recovery Medium
a. Carefully resuspend the cells in each well of the Nucleocuvette™ with 70 µl of pre-warmed growth medium, and
mix gently by pipetting up and down (~3 times) to ensure an even distribution of cells in suspension.
2.8 Plate Cells
a. Retrieve the two 24-well cell culture plates prepared in step 2.1.
b. Of the 100 µl cell suspension (for each reaction), transfer 50 µl to the first pre-warmed 24-well plate for genomic
analysis. Transfer the other 50 µl to the second pre-warmed plate for assays/clonal expansion.
c. Incubate the cells in humidified 37°C/5% CO2 incubator.
d. Replace media after 24 hours.
Note: Maintain the second plate by replacing medium and splitting as necessary until clonal expansion, assays, or
banking.

9
USER MANUAL
3. Post-Nucleofection
a. 48 hours after transfection, extract genomic DNA from one plate for genomic analysis. Amplify the DNA region
around the targeted site from experimental and control cells and submit the amplicons for Sanger sequencing.
After sequencing, we recommend analyzing the amplicons using Synthego’s Inference of CRISPR Edits (ICE)
online tool. See Synthego’s Knockout Analysis protocol for instructions on how to extract DNA, perform PCR,
and prepare the amplicons for Sanger sequencing. This protocol also contains instructions for how to analyze
editing efficiency using the ICE tool and how to interpret ICE results.
b. Maintain the second plate by replacing medium and splitting as necessary until clonal expansion, assays,
or banking. For instructions on how to clone knockout cells, see Synthego’s Limiting Dilution and Clonal
Expansion protocol. For Western Blot analysis, we recommend assessing protein quantity for a time course
of at least 7 days and use a negative control.
Lipofection Protocol
This protocol is meant to serve as a starting point for lipofection of immortalized cells. It may be necessary to
experimentally optimize volumes and ratios for RNP formation for each cell type and for other culture plate formats.
It is critical to add reagents in the order recommended below. Prepare the RNP complexes with the Lipofectamine™
Cas9 Plus™ Reagent and Opti-MEM™ I Reduced Serum Medium in a separate tube (Tube 1) before adding diluted
Lipofectamine™ CRISPRMAX™ Reagent (Tube 2). Volumes are for each reaction and should be scaled up proportionally
to the number of desired reactions.
Important Considerations
• All Synthego and CRISPRMAX™ reagents should be stored according to the manufacturer’s recommendations.
• This protocol was optimized in HEK293 cells and can be used for other common cell lines such as A549, U2OS,
HeLa, CHO, and MCF-7.
• Successful transfection is critically dependent on cell density. It may be necessary to optimize cell seeding densities
in order to determine the most appropriate level of confluence for transfection.
• Cell seeding is based on the rate of cell growth. For fast growing cells, seed fewer cells. Suggested starting cell
numbers are listed in the protocol below.
• In order to maximize CRISPR editing of adherent cells, be sure to trypsinize the cells prior to lipofection (Step 2.5b).
• Use cells at lowest passage number possible.
• Cas9 nuclease can be diluted in Opti-MEM™ I Reduced Serum Medium in order to achieve a working concentration
according to the plate volume.
• Synthego recommends sgRNA:Cas9 ratio of 1.3:1 for RNP formation. It may be necessary to optimize ratios for
different cell lines/conditions.
• RNP complexes are formed in Opti-MEM™ I Reduced Serum Medium and can be added directly to cells in culture
medium irrespective of antibiotics. Following transfection, it is not necessary to remove RNP complexes or add/
change medium.

10
USER MANUAL
Material Ordering Information
Positive control multi-guide sgRNA (optional) Recommended:Transfection Optimization Kit

(Synthego, available at checkout)
Transfection control (optional) Recommended: pMAXGFPTM (Lonza)
Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (includes Thermo Fisher Scientific, Catalog #CMAX00001
Cas9 Plus Reagent and CRISPRMAX™ Transfection Reagent)
Opti-MEMTM I Reduced Serum Medium Thermo Fisher Scientific, Catalog #31985062
Normal growth medium Cell-type dependent
24-well cell culture plates Corning, Catalog #3526
TrypLE Express or preferred cell dissociation reagent Multiple vendors (e.g., Thermo Fisher Scientific)
1X PBS, cell culture grade Multiple vendors (e.g., Thermo Fisher Scientific)
Cell counter Multiple vendors (e.g., Thermo Fisher Scientific)
Microcentrifuge tubes Multiple vendors (e.g., Eppendorf)
1. Pre-Lipofection
1.1 Seed cells
a. Seed cells and incubate in 37°C/5% CO2 incubator overnight so that they are 30-70% confluent on the day of
transfection (may take 1-2 days).
2. Setup & Lipofection

2.1 Prepare Destination Plates
a. Pre-warm 1 ml of normal growth medium in each well of two 24-well cell culture plates. After lipofection, the
cells will be split into two wells on the duplicate plates. The cells on the first plate will be lysed and processed to
analyze editing efficiency. The cells on the second plate will be cultured for use in assays, banking, and/or single-
cell cloning. Consider whether you plan to culture control cells on the second plate.
2.2 Assemble RNP Complexes (1.3:1 sgRNA to Cas9 ratio)
a. Dilute multi-guide sgRNA and Cas9 to 3 μM working stock concentrations (3 pmol/μl).
Note: If you followed the Gene Knockout Kit v2 QuickStart Guide, you may have 30 μM stock concentrations (30 pmol/μl), so
be sure to dilute further to 3 μM (3 pmol/μl).

11
USER MANUAL
b. Prepare RNPs in microcentrifuge tube (Tube 1). Use the quantities (per reaction) in the table below.
Note: You may need to experimentally determine the optimum amounts of sgRNA and Cas9 nuclease. Synthego
recommends a ratio of 1.3:1 sgRNA to Cas9 for RNP formation.
c. Incubate RNPs for 5-10 minutes at room temperature.
2.3 Prepare Transfection Solution

a. In a separate microcentrifuge tube (Tube 2), dilute Lipofectamine™ CRISPRMAX™ Reagent in Opti-MEM™ I
Reduced Serum Medium. Use the quantities (per reaction) in the table below.
b. Incubate transfection solution for 5 minutes at room temperature.
2.4 Prepare RNP-Transfection Solution
a. Add the transfection solution (Tube 2) directly to RNPs (Tube 1), and mix well by pipetting up and down.
b. Incubate for 5-10 minutes at room temperature. Do not exceed 30 minutes.
Note: Synthego highly recommends reverse transfection (RNPs are added to wells first and cells are added second), as
this method has resulted in high editing efficiencies.
RNP Preparation (Tube 1)
Component Molarity Volume (per reaction)
Opti-MEM™ I Reduced Serum Medium - 25 μl
Multi-guide sgRNA 3 μM (pmol/μl) 1.3 μl (3.9 pmol)
Cas9 3 μM (pmol/μl) 1 μl (3 pmol)
Lipofectamine™ Cas9 Plus Reagent - 1 μl
Total volume - 28.3 μl
2.5 Prepare Cells

Note: For suspension cells, re-suspend in growth medium and mix well. Skip steps a and b below and proceed
to step c.
a. Wash cells with 1X PBS (enough to cover bottom of each well), then aspirate PBS.
Transfection Solution (Tube 2)
Reagent Volume (per reaction)
Opti-MEM™ I Reduced Serum Medium 25 μl
Lipofectamine™ CRISPRMAX™ Transfection Reagent 1.5 μl
Total volume 26.5 μl

12
USER MANUAL
b. Add TrypLE Express or preferred cell dissociation reagent (enough to cover bottom of each well), incubate
for 5 minutes in a humidified 37°C/5% CO2 incubator. Resuspend cells in an equivalent volume of growth
medium to stop the trypsin reaction.
c. Count cells to determine density.
d. Transfer 0.42 – 1.2 x 105 cells per reaction to a microcentrifuge tube.
e. Centrifuge cells at 200 x g for 5 minutes.
f. Resuspend cells in 500 μl of the growth medium.
2.6 Transfect Cells
a. Add the RNP-transfection solution to wells of one of the 24-well cell culture plates prepared in step 2.1 (see
table below).
b. Add the cell suspension in growth medium to the wells (see table below). Mix well.
c. Retrieve the second 24-well cell culture plate prepared in step 2.1.
d. Of the 550 µl cell suspension (for each reaction), transfer 225 µl to the duplicate pre-warmed 24-well plate.
There are now duplicate plates: one for genomic analysis and one for assays/clonal expansion
e. Incubate the cells in humidified 37°C/5% CO2 incubator for 2-3 days
f. Replace media after 24 hours.
Note: Maintain the second plate by replacing medium and splitting as necessary, until clonal expansion, assays, or
banking.
3. Post-Lipofection
RNP-Transfection Solution & Cell Suspension
Reagent Volume (per reaction)
RNP-Transfection Solution 50 μl
Cell suspension in growth medium 500 μl
Total volume 550 μl
a. 48 hours after transfection, extract genomic DNA from one plate for genomic analysis. Amplify the DNA region
around the targeted site from experimental and control cells and submit the amplicons for Sanger sequencing.
After sequencing, we recommend analyzing the amplicons using Synthego’s Inference of CRISPR Edits (ICE)
online tool. See Synthego’s Knockout Analysis protocol for instructions on how to extract DNA, perform PCR,
and prepare the amplicons for Sanger sequencing. This protocol also contains instructions for how to analyze
editing efficiency using the ICE tool and how to interpret ICE results.
b. Maintain the second plate by replacing medium and splitting as necessary, until clonal expansion, assays, or
banking. For instructions on how to clone knockout cells, see Synthego’s Limiting Dilution and Clonal Expansion
protocol. For Western Blot analysis, we recommend assessing protein quantity for a time course of at least 7
days and use a negative control.

13
USER MANUAL
Troubleshooting Guide
Nucleofection
Problem Possible Cause(s) Recommended Solutions
Loss of cells during pelleting/removing

Use caution when aspirating supernatant.
supernatant before Nucleofection
No cells on plate
After Nucleofection, add medium to the cuvette and pipette up and
Cells left in Nucleofection cuvette down multiple times to ensure cells are not left in the bottom of the
cuvette.
Ensure that the cell suspension is mixed thoroughly before adding to

Non-uniform cell suspension pre-complexed RNPs, and continue to gently agitate the suspension
to avoid settling.
Uneven distribution of
cells between reactions Cells left in Nucleofection cuvette
After Nucleofection, add medium to the cuvette and pipette up and
down multiple times to ensure cells are not left in the bottom of the
Cells not mixed enough after cuvette and that the concentration of cells is uniform.
Nucleofection
Cells should be viable and in culture for several passages. Avoid

Cell culture conditions were suboptimal excessive cell densities or high cell confluencies as this may
decrease cell viability post Nucleofection.
Avoid harsh conditions during cell harvesting, especially

Cells were damaged by harvesting
Low viability centrifugation at high speed or overexposure to trypsin. Pipette cells
procedure or through handling
smoothly.
Transfer cells immediately into pre-warmed medium as

Cells were in Nucleofector Solution too
recommended in the optimized protocol. Avoid leaving cells in
long
Nucleofector Solution for longer than 15 minutes.
The optimal number of cells per transfection reaction varies

Cell number between cell types. Optimize cell number using the GFP plasmid
provided in the 4D Nucleofector Kit.
Low transfection
efficiency Use the recommended Lonza Nucleofector solution and program
Wrong Nucleofector Solution/program for your cell type. If Lonza does not have an optimized protocol and
for cell type established solution for your cell type, it is recommended to do an
transfection optimization using GFP plasmid prior to editing.
Forward transfection We recommend reverse transfection for maximum efficiency.
Low editing efficiency

Optimize ratio of sgRNA:Cas9 using a positive control multi-guide
sgRNA:Cas9 ratio
sgRNA (e.g., TRAC) in your cell type.

14
USER MANUAL
Lipofection
Problem Possible Cause(s) Recommended Solutions
Loss of cells during pelleting/removing

No cells on plate Use caution when aspirating supernatant.
supernatant before Lipofection
Cells should be viable and in culture for several passages. Avoid

Cell culture conditions were suboptimal excessive cell densities or high cell confluencies as this may
decrease cell viability post lipofection.
Low viability
While increasing the amount of Lipofectamine™
Too much Lipofectamine™ reagent may improve overall transfection and editing efficiency, it
may also decrease the viability of the cells.
Low transfection The optimal number of cells per transfection reaction varies
Cell number
efficiency between cell types. Optimize cell number using the GFP plasmid.
Test different volumes of LipofectamineTM using a positive control

Volume of Lipofectamine™
multi-guide sgRNA (e.g., TRAC) in your cell type.
Test different concentrations using a positive control multi-guide

Concentration of multi-guide sgRNA
Low editing efficiency
Forward transfection We recommend reverse transfection for maximum efficiency.
Optimize ratio of sgRNA:Cas9 using a positive control multi-guide

sgRNA:Cas9 ratio
Additional Information About Synthego

For an up-to-date list of all Synthego Synthego is the leading genome engineering
Protocols and other resources, innovation company. The company’s
please visit Synthego.com/resources. automated, full stack genome engineering
platform enables broader access to CRISPR
For technical assistance,
to accelerate basic scientific discovery,
contact our Scientific Support Team:
uncover cures for diseases, and develop novel
Ph: 888.611.6883 Email: support@synthego.com synthetic biology applications. Headquartered
in Silicon Valley, Synthego is used by scientists
from the largest global biotechnology
companies and global biology universities to
unlock the potential of gene editing.

15

GKO v2 User Manual

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

GKO v2 User Manual

Uploaded by

Copyright:

Available Formats

USER MANUAL

Gene Knockout Kit v2

Gene Knockout Kit v2 Synthego.com/contact

Additional Materials Required 4

Read complete user manual before using the kit.

Gene Knockout Kit v2 Synthego.com/contact

stranded break (DSB) in the DNA (denoted by the scissors).

Target DNA PAM

Figure 2. Synthego's multi-guide design.

+ For details on Synthego’s money-back guarantee, please visit Synthego.com/legal/money-back-guarantees.

Gene Knockout Kit v2 Synthego.com/contact

21+ bp fragment deletions).

1.5 ml Nuclease-free water - Room temperature

Additional Materials Required

Transfection Optimization Kit Includes: Synthego (available at checkout)

Gene Knockout Kit v2 Synthego.com/contact

Transfection Using Positive Control Multi-guide sgRNA

4 DAYS PCR & Sanger Sequencing

Target Knockout Experiment

Transfection Using Target Multi-guide sgRNA

4 DAYS PCR & Sanger Sequencing

Clonal Isolation of Knockout

Gene Knockout Kit v2 Synthego.com/contact

Control Description Purpose

Gene Knockout Kit v2 Synthego.com/contact

Additional Materials Required

Positive control multi-guide sgRNA (optional) Recommended:Transfection Optimization Kit

Transfection control (optional) Recommended: pMAXGFPTM (Lonza)

Normal growth medium Cell-type dependent

24-well cell culture plates Corning, Catalog #3526

Cell counter Multiple vendors (e.g., Thermo Fisher Scientific)

Microcentrifuge tubes Multiple vendors (e.g., Eppendorf)

4D-Nucleofector™ System (4D-Nucleofector™ Core Unit and Lonza

Cell specific Lonza 4D-Nucleofector™ X Kit with 16-well Lonza

Gene Knockout Kit v2 Synthego.com/contact

2. Setup & Nucleofection

Nucleofector™ Solution 24.6 µl 25 µl 24 µl 18 µl 18 µl

pmaxGFP vector 0.4 µl - - - -

Cas9 2NLS nuclease - - 1 µl 1 µl 1 µl

Gene Knockout Kit v2 Synthego.com/contact

2.5 Prepare Cell/RNP Solution

Gene Knockout Kit v2 Synthego.com/contact

Gene Knockout Kit v2 Synthego.com/contact

Additional Materials Required

Material Ordering Information

Positive control multi-guide sgRNA (optional) Recommended:Transfection Optimization Kit

Transfection control (optional) Recommended: pMAXGFPTM (Lonza)

Opti-MEMTM I Reduced Serum Medium Thermo Fisher Scientific, Catalog #31985062

Normal growth medium Cell-type dependent

24-well cell culture plates Corning, Catalog #3526

Cell counter Multiple vendors (e.g., Thermo Fisher Scientific)

Microcentrifuge tubes Multiple vendors (e.g., Eppendorf)

2. Setup & Lipofection

Gene Knockout Kit v2 Synthego.com/contact

2.3 Prepare Transfection Solution

RNP Preparation (Tube 1)

Component Molarity Volume (per reaction)

Opti-MEM™ I Reduced Serum Medium - 25 μl

Multi-guide sgRNA 3 μM (pmol/μl) 1.3 μl (3.9 pmol)

Cas9 3 μM (pmol/μl) 1 μl (3 pmol)

Lipofectamine™ Cas9 Plus Reagent - 1 μl

Total volume - 28.3 μl

2.5 Prepare Cells