Pharma Starts with You

CRISPR Gene Editing Workflow
A toolbox for successful gene editingin drug discovery and development

A VERSATILE
AND EFFICIENTWORKFLOW

The power to edit a gene is the power to change its function, and withit the biology of a cell. From generating novel cell lines and better animalmodels for the discovery and preclinical phases of therapeutic researchto actually creating a therapeutic itself, such as CAR T-cell therapies,
CRISPR gene editing is allowing science to advance rapidly. The
downstream applications are countless and are changing the way weperform R&D and the way we think about treating and curing disease.
We present a workflow that guides you through the four main stepsof gene editing using the cancer cell line PC3. We designed a geneknockout strategy that uses knock-in of GFP and the puromycin
resistance gene to disrupt ICAM-1, a gene known to play a role incell adhesion and cell signaling. GFP and puromycin resistance serve
as easy to follow markers for successful gene disruption. Although
we chose a knock-in strategy, this workflow can be applied to mostother gene editing experiments. Follow us through the generationand downstream analysis of ICAM-1 knockout cell lines and see how
Bio-Rad products support and accelerate gene editing at each stage.

2 | BIO-RAD LABORATORIES, INC.

1
2
3
4

TRANSFECTION

GENE PULSER Xcell™
ELECTROPORATOR

TransFectin™
LIPID REAGENT

S3e™ CELL SORTERZOE ™ CELL IMAGER

V3 WESTERNWORKFLOW ™

CFX REAL-TIME PCR SYSTEM

CFX AUTOMATION SYSTEM II

BIO-PLEX® 200 SYSTEMENRICHMENT AND
SINGLE-CELL ISOLATIONCONFIRMATION OF EDITSQX200™ DROPLET DIGTAL™
PCR SYSTEMDOWNSTREAM ANALYSIS

BIO-RAD LABORATORIES, INC. | 3

A VERSATILE AND EFFICIENT WORKFLOW

BIO-RAD’S CRISPR GENE EDITING WORKFLOW:
GENERATING AND ANALYZING ICAM-1 KNOCKOUT CELL LINESTRANSFECTION

TRANSFECT WITH TRUSTED TOOLSGENE PULSER Xcell™
ELECTROPORATORZOE™ CELL IMAGER

TransFectin™
LIPID REAGENT

TC20™ CELL COUNTER

After you have designed your gene editing strategy, the firststep in the CRISPR gene editing workflow is to identify the best
method for delivering the CRISPR-Cas9 system into your cellsof interest, be they animal or human, tumor-derived or wild
type. Transfer efficiency and subsequent cell viability are very
important when considering which method to use. Bio-Rad
provides several transfection options.
The industry-leading Gene Pulser Xcell Electroporation System
has been used in over 700 peer-reviewed studies and has
supported a wide range of research for more than 20 years.
If lipid-mediated transfer proves most efficient, TransFectin Lipid
Reagent provides a gentle, effective option even for primary celllines. The ZOE Fluorescent Cell Imager can be used to monitor
transfection efficiencies and cell health right on your bench, andthe TC20 Automated Cell Counter automates cell counting at
multiple stages of transfection, improving accuracy in cell counting
across all stages of the experiment.
We designed a knockout strategy that uses knock-in of adonor template containing the GFP and puromycin resistancegenes to disrupt ICAM-1. PC3 cells were transfected using
either TransFectin Reagent or the Gene Pulser Xcell System.
GFP fluorescence was used as an initial readout forsuccessful transfection.

SUCCESS STORY CRISPR RNP Electroporation of Zygotes (CRISPR-EZ)
uses the Gene Pulser Xcell System and enables highly efficient highthroughput in vivo genome editing (Chen S et al. 2016). CRISPR-EZ is a
simple, economical, high-throughput, and highly efficient method withthe potential to replace microinjection for in vivo genome editing.
Chen S et al. (2016). Highly efficient mouse genome editing by CRISPR ribonucleoprotein
electroporation of zygotes. J Biol Chem 291, 14,457–14,467.

4 | BIO-RAD LABORATORIES, INC.

TRANSFECTION
TRANSFECTION

A

B

Optimizing transfection efficiency. PC3 cells (ATCC) were cultured
in Ham’s F-12 Nutrient Mix (GIBCO) with 10% HyClone FBS (GEHealthcare), 1% Penicillin-Streptomycin (GIBCO) and maintained
at 37°C and 5% CO2 for 7 days. Prior to transfection, cells werecounted using the TC20 Cell Counter. One of two different CRISPRCas9 plasmids targeting ICAM-1 (OriGene) and a donor vector
containing left and right homologous arms as well as a GFP-Puro
functional cassette (OriGene) were transfected into PC3 cells
using either the Gene Pulser Xcell Electroporation System or lipidmediated transfection with TransFectin Lipid Reagent. Media of bothelectroporated and TransFectin treated cells was replaced with fresh
growth media 24 hours after treatment. The ZOE Fluorescent Cell
Imager was used to assess GFP expression in transfected culturesand showed that the plasmids had been successfully transferred into
cells. Merged (A) and GFP channel (B) view of PC3 cells 24 hours
after transfection using the Gene Pulser Xcell System captured using
the ZOE Fluorescent Cell Imager. TransFectin and the Gene Pulser
Xcell System provided similar levels of transfection efficiency(data not shown).

BIO-RAD LABORATORIES, INC. | 5

ENRICHMENT AND

SINGLE-CELL

ISOLATION
SORT CELLS AND SAVE 30 DAYS

S3e™ CELL SORTERZOE™ CELL IMAGER

Once you have transfected your cells you need to enrich for yourcells of interest. This step speeds up your workflow and reduces
unnecessary spending on reagents and consumables by allowing
identification and expansion of only those cells that carry desirededits. Enrichment also reduces the number of passages cells
undergo, ensuring healthy cells for downstream analysis. With the
S3e Cell Sorter and ZOE Fluorescent Cell Imager, enrichment iseasily performed at your bench without the need for a core facility
and your workflow can be shortened by as much as 30 days.
We enriched for successfully transfected cells by sorting GFPpositive PC3 cells using the S3e Cell Sorter. After enrichment andsubsequent puromycin selection, the S3e Cell Sorter was used toisolate single GFP-positive, puromycin resistant cells for subsequent
expansion. The ZOE Cell Imager allowed early confirmation of
successful enrichment pre- and post-puromycin treatment.

SUCCESS STORY Whether you are analyzing difficult-to-interpret heterogeneous
cell populations or are trying to isolate edited cells for expansion and creationof homogeneous cell lines, reliable single-cell sorting is a powerful andimportant first step. Bio-Rad bulletin 6853 demonstrates a simple four-step
workflow that uses the TC20™ Cell Counter and ZOE Cell Imager to verify thereliability of single-cell sorting on the S3e Cell Sorter.
Visit bio-rad.com/CRISPR1 to read more.

6 | BIO-RAD LABORATORIES, INC.

14

TRANSFECT14

ENRICHDAYSTRADITIONAL WORKFLOW

28

TRANSFECTDAYSIsolate only GFP-positive cells

DAYS

7
7
14
DAYS

PUROMYCIN
SELECTIONDAYSPOST PUROMYCIN
SELECTION

7
35

PUROMYCIN
SELECTIONDAYS+POST PUROMYCIN
SELECTIONDAYSSINGLE-CELLSORTING

DAYS

56
TOTAL

DAYS

14

MANUAL SINGLE-CELL
ISOLATION OR DILUTION

DAYS

84
TOTAL

+

DAYS

14

DAYS POST
PUROMYCIN
ADDITION

14

DAYS POST
PUROMYCIN
ADDITION

Accelerated target cell enrichment. Cells transfected either by electroporation or by lipid-mediated TransFectin Reagent were
cultured for 14 days. Live GFP-positive cells were sorted from a mixed cell culture using the S3e Cell Sorter. Fourteen days later
enriched cells were subjected to puromycin selection to further select for edited cells. Post-treatment, single GFP-positive cells were
sorted using the S3e Cell Sorter. Single-cell isolation and GFP fluorescence were confirmed using the ZOE Cell Imager. Cells were thenpassaged for 2 weeks until 80% confluent for further analysis. This accelerated workflow allowed generation of confluent cultures ofclonal ICAM-1 knockout cell lines in 56 days instead of the more than 84 days.

BIO-RAD LABORATORIES, INC. | 7

ENRICHMENT AND SINGLE-CELL ISOLATION

BIO-RAD’S WORKFLOW

CONFIRMATIONOF EDITSCONFIRM EDITS WITH CONFIDENCE

V3 WESTERN WORKFLOW ™

ZOE™ CELL IMAGER

QX200™ DROPLET DIGITAL™
PCR SYSTEM

CFX REAL-TIME PCR
DETECTION SYSTEM

NHEJ

WT

Once you have enriched your cells of interest, you need to confirm that you
have successfully edited your target cells before moving on to downstream
assays. This can be accomplished through direct detection of edits
using genomic methods or through indirect detection using cellular orproteomic methods.
PCR-based methods provide precise and sensitive detection andquantification of edits. High-fidelity PCR, Droplet Digital PCR (ddPCR™),
real-time PCR, and high resolution melt analysis (HRMA) allow analysis
of NHEJ and HDR edits. The CFX Real-Time PCR Detection System
is a versatile platform for quantitative PCR and HRMA. For standard
PCR and sequencing validation, iProof™ High-Fidelity PCR Reagentsminimize the incorporation of errors. When editing efficiencies are low,
ddPCR technology allows identification of allele changes with unmatchedprecision; mutations present at frequencies ≤0.5% can be identified.
The ZOE Fluorescent Cell Imager allows confirmation of knocked in
and knocked out protein expression in your newly edited cells right at
your bench. The V3 Western Workflow quantitatively measures protein
expression faster and with more accuracy than other western blot
procedures. By incorporating validated PrecisionAb™ Western Blotting
Antibodies, you can further remove uncertainty from your experiments.
We used the ZOE Cell Imager and V3 Western Workflow to confirm successful
gene edits. These orthogonal methods, along with direct methods such as
Droplet Digital PCR or HRMA using qPCR (data not shown), give you theconfidence to move forward with downstream analysis of your edited cells.
SUCCESS STORY Bio-Rad bulletin 6712 describes a rapid, cost-effective, andhigh-throughput Droplet Digital PCR method that provides convenient readout
for the detection of edited alleles and for the optimization of CRISPR andTALEN editing protocols. This method is especially useful for those working
with primary and pluripotent stem cells, where editing frequency is often low.
Visit bio-rad.com/CRISPR2 to read more.

8 | BIO-RAD LABORATORIES, INC.

CONFIRMATION OF EDITS

A

B

Confirmation of successful gene edits byimmunocytochemistry. Control and edited cells were plated
onto microscope slides 48 hours prior to staining with antiICAM-1 antibody (red), and DAPI (blue)-containing mounting
medium. Slides were imaged at the bench using the ZOECell Imager. Edited cells show expression of GFP but no
ICAM-1 expression. A, immunostaining of control PC3 cells
reveals abundant ICAM-1 expression. B, CRISPR-edited PC3
cells have lost ICAM-1 expression and gained stable GFP
expression.

Confirmation of successful gene editing by
western blot. Control and edited cells were lysed on
ice in RIPA Buffer (VWR) containing protease inhibitor
cocktail (VWR), sonicated, and spun down to remove
particulates. Lysates were centrifuged again andthe supernatant was diluted in 4x Laemmli Sample
Buffer (cat. #1610747) containing b-mercaptoethanol(cat. #1610710) and run on a 12% acrylamide
precast stain-free gel (cat. #3451012). Proteins weretransferred to nitrocellulose for 7 minutes at 100 V
using the Trans-Blot® Turbo™ Transfer System(cat. #1704150). The blot was blocked with 1x
Tris buffered saline (TBS) with 1% casein and thenrinsed with 1x phosphate buffered saline Tween 20.
CD54 PrecisionAb™ Primary Antibody recognizing
ICAM-1 (cat. #VPA00014K), Goat Anti-Rabbit IgGStarBright™ Blue 700 (cat. #12004161) (red), AntiGAPDH hFAB™ Rhodamine Labeled Primary Antibody(cat. #12004167) (blue), and Anti-turboGFP Primary
Antibody (OrigGene) (green) were applied. The blot
was imaged on the ChemiDoc™ MP Imaging System(cat. #17001402).

BIO-RAD LABORATORIES, INC. | 9

DOWNSTREAMANALYSISANALYZE AND MAKE DISCOVERIESZE5™ CELL ANALYZER

CFX AUTOMATION SYSTEM II

BIO-PLEX 200 SYSTEM®

V3 WESTERN WORKFLOW ™

QX200™ DROPLET DIGITAL™
PCR SYSTEM

Now that you have confirmed that your cells are correctly edited, you can begin
to investigate their phenotype. You may even wish to further perturb your systemwith a drug as part of a cell-based assay during target or lead discovery andvalidation. Bio-Rad provides tools to study your newly generated cells lines at
the genomic, cell, and proteomic levels.
We provide predesigned and validated, as well as customizable, tools for highthroughput gene expression analysis, including the CFX Automation System,
PrimePCR™ Gene Expression Assays and Panels, and CFX Maestro™ Softwarefor Bio-Rad real-time PCR detection systems. For more sensitive absolute
quantitation, use predesigned or custom ddPCR™ Genome Edit Detection
Assays and our Droplet Digital PCR Systems.
The ZE5 Cell Analyzer allows you to study the effects of your edits at thecellular level. This flexible analyzer can be run by novices and experts alike.
It accommodates up to 30-parameter experiments. This high-level multiplexing
allows you to more fully characterize your edited cells and their response todifferent assay conditions.
Multiplex proteomic analysis using the V3 Western Workflow and validated
Bio-Plex Multiplex Immunoassays gives you a complete picture of the proteomic
profile of your new cell lines. Validated PrecisionAb™ Western Blotting Antibodies
and disease-specific or customizable multiplex Bio-Plex Assays ensure sensitiveand reliable results.
To characterize our ICAM-1 knockout cells we used PrimePCR Gene Expression
Panels and CFX Maestro Software to assess changes in gene expression.
SUCCESS STORY In this brochure we have shown that incorporating flow cytometry into
our CRISPR Gene Editing Workflow speeds time to results. Bio-Rad bulletin 6961
provides expanded experimental details, as well as additional results from our ICAM-1
knock out study.
Visit bio-rad.com/CRISPR3 to read more.

10 | BIO-RAD LABORATORIES, INC.

DOWNSTREAM ANALYSIS

PC3 + TNF-a
PC3
Edited PC3 + TNF-a
Edited PC3

A

27
Edited PC3 + TNF-a, normalized expression

ITGA7
ITGB1
COL7A1
CD44
COL5A1
KAL1
ITGA1
MMP15
COL12A1
THBS3
ITGB5
SPG7
MMP16
LAMB1
ADAMTS1
ITGB4
TIMP2
ITGA6
CTNND2
CDH1
VCAN
ITGAV
MMP14
LAMB3
COL6A2ECM1ITGA2
COL6A1
MMP10
ICAM1
MMP1
MMP13
TIMP1

B

GENE

9

25

1

SPP1

23

2

SELL

3

VCAN

4

LAMB3

5

MMP10

6

MMP1

7

SGCE

14
13

8

MMP13

12

9

ITGB1

10

LAMB1

11

ADAMTS1

2–11

12

SPARC

2–13

13

ECM

14

ITGB4

2

1

5 6

2–1
2–3
2

7

8

4

–5

11

2–7
12 3

2–9

10

2–15
2

–16

2

–13

2
2
2
2
2
PC3 normalized expression
–10

–7

–4

–1

2

2

5

Relative normalized
expression

C
5
4
3
2
1
0
COL1A1

CTGF

ECM1

FN1

ITGA1

ITGA3
ITGB2
Target

MMP11

MMP15

SPARC

THBS3

TIMP2

Gene expression analysis in control and edited cells in response to TNF-a treatment. Control and edited
cells were treated with TNF-a to stimulate ICAM-1 expression. Cells were lysed using the SingleShot™ Cell Lysis
Kit (cat. #1725080), which allows preparation of gDNA-free RNA directly from cell culture. SingleShot Cell Lysates
were added to three different PrimePCR Predesigned 384-Well Pathway Plates: Male Urogenital Diseases Tier 1,
Extracellular Matrix, and Neoplasm Metastasis. We screened 962 targets by real-time PCR to look for changes ingene expression using a CFX384 Touch™ Real-Time PCR Detection System (cat. #1855485) and analyzed resulting
data using CFX Maestro Software (cat. #12004110 ). A, relative normalized expression plot shows little or no change
in expression of ICAM-1 in edited cells compared to wild-type PC3 in response to TNF-a treatment. These results
also show that edited cells retained a low level of ICAM-1 expression. B, a scatter plot provides an alternate view ofchanges in expression. The red line represents a 4-fold increase and the green line a 4-fold decrease in expression
compared to control cells (unstimulated wild-type PC3 cells); C, interestingly other cell adhesion markers showedaltered expression between wild-type and edited PC3 cells before and after stimulation with TNF-a, suggestingthat cells are using these gene products to compensate for lack of a ICAM-1. PC3 ( ); PC3 + TNF-a ( ); edited
PC3 ( ); edited PC3 + TNF-a ( ).

BIO-RAD LABORATORIES, INC. | 11

WHY BIO-RAD?
Bio-Rad has over 60 years experience supporting life science
research and clinical diagnostics. We are the leader in digital
PCR, transfection, and in gene and protein expression analysis
technologies. Our innovative products are available as standalone solutions or combined into workflows that are optimized
to minimize downtime and to provide the right answer the firsttime, every time. The CRISPR Gene Editing Workflow is only oneof many Bio-Rad workflows designed to shorten discovery anddevelopment time lines for novel therapeutics.

Cells
S3e Cell SorterZOE Fluorescent Cell Imager
Gene Pulser Xcell Electroporation Systems
TransFectin Lipid Reagent
Helios® Gene Gun Systems
TC20 Cell Counter
Antibodies for Flow Cytometry and Immunohistochemistry
Genes
QX200 Droplet Digital PCR System
PrimePCR Assays for qPCR and ddPCR
CFX Real-Time PCR Detection Systems
CFX Maestro Sofware
Hard-Shell® PCR Plates
C1000 Touch™ Thermal Cycler
Ready Gel® Precast Agarose Gels
SsoAdvanced™ Universal Supermixes
Precision Melt Supermix
iProof High-Fidelity PCR Reagents
Precision Melt Analysis™ Software
Proteins
Bio-Plex Multiplex Immunoassay Systems
Bio-Plex Multiplex Immunoassays
Stain-Free Mini and Midi Protein Gels
Trans-Blot Turbo Transfer System
ChemiDoc MP Imaging System
PrecisionAb Validated Western Blotting Antibodies
Antibodies for Flow Cytometry and ImmunohistochemistryProtein Sample Preparation Beads and Kits

As part of our commitment to your research, we provide:
Products and reagents that are reliable and validated
Responsive, trusted global service and technical support teams■■ Technical expertise across multiple workflows and applications■■■■

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Enrichment

Confirmation

Analysis

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Visit bio-rad.com/web/CRISPRinfo to learn more about our CRISPR toolkit.
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Bulletin 6947 Ver A

US/EG

17-04290617 Sig 1216