Fast and Efficient Workflow to Monitor CRISPR-Cas9 Gene
Editing of Haploid Cell Lines
Laura Moriarty, PhD; Meiye Wu, PhD; and Elizabeth Jordan, PhD
Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA

Drug Discovery and Development

Bulletin 7079

Abstract

A great deal of excitement surrounded the introduction of CRISPR as a simplified gene editing
technique. Its speed, low cost, and ability to be used in many model systems redefine what
is possible in drug discovery and development. However, current workflows for generatingknockout cell lines using CRISPR-Cas9 can be complex and inefficient. In this study, wedemonstrate a novel workflow that includes Droplet Digital™ PCR, high resolution melt analysis,
western blotting, and real-time PCR to generate and validate a knockout haploid cell line in lessthan five weeks.
Introduction
CRISPR-Cas9 is the simplest gene editing technique discoveredto date. Based on the adaptive immune system of bacteria andarchaea, the CRISPR-Cas9 system uses short RNAs to direct
Cas9 nuclease to target DNA sequences. Because it uses RNA
rather than a protein to target nuclease activity, CRISPR-Cas9
can be retargeted by simply synthesizing or ordering a new
guide RNA (gRNA), which also makes this technique amenable
to high order multiplexing. These attributes make CRISPR-Cas9
ideal for introducing mutations in protein-encoding genomic
DNA regions, which allows protein function to be studied in its
native cellular context.
In this study, we report a novel workflow that incorporates
Droplet Digital PCR (ddPCR™), high resolution melt (HRM)
analysis, western blotting, and RT-PCR to generate Sanger
sequencing validated knockout haploid human cell lines. For
this experiment we targeted the BUB1B gene, which encodes a
kinase involved in spindle checkpoint function and is implicatedin cancer, as the loss or reduced function of the spindle
checkpoint has been found in many forms of cancer. The
knock out or knock down of genes implicated in cancer is oneof the many ways gene editing is being used to further study
disease, as it allows researchers to model the effects of different
mutations.
Our Haploid Gene Editing Workflow (Figure 1) combines multiple
techniques to provide edited cells in less than five weeks byallowing easy assessment of transfection protocols and quickconfirmation of edits, and through the inclusion of validated tools
to analyze both gene and protein expression. By using haploidcells in this workflow we were able to simplify the workflow
and associated data analysis because only a single inactivatedallele is required to generate a loss-of-function phenotype.
The resulting edited haploid cell lines can be used in many
downstream assays such as target identification, mechanism ofaction studies, and cell-based toxicity assays.

Population-level screening

Clonal screening

Protein expression confirmation

Gene expression analysis

Droplet Digital PCR

CFX Automation System II

V3 Western Workflow™

CFX Maestro™ Software

Fig. 1. Haploid Gene Editing Workflow.

Fast and Efficient Workflow to Monitor CRISPR-Cas9 Gene Editing of Haploid Cell LineseHAP-1 cells (Horizon Discovery) were transfected withCRISPR-Cas9 targeting BUB1B. The clones were sent out
for Sanger sequencing to confirm edits.
Non-Homologous End Joining (NHEJ) Assay

The ddPCR NHEJ Genome Edit Detection Assay specificfor our BUB1B mutation was designed on bio-rad.com/
digital-assays (catalog #12002314). The assay was run on
the QX200™ ddPCR System and the fractional abundanceof NHEJ DNA was determined using QuantaSoft™ Analysis
Pro Software as described in the ddPCR NHEJ Genome Edit
Detection Assay protocol (#10000065278).
High Resolution Melt (HRM) Assay

A high resolution melt assay was designed and run as per
the Precision Melt Analysis™ Software Instruction Manual(#10000080911). Briefly, cells were lysed using the SingleShot™
Cell Lysis Kit (catalog #1725080) to generate gDNA directlyfrom cell culture, and HRM analysis was performed using
Precision Melt Supermix for High Resolution Melt Analysis(catalog #1725110) on the CFX384 Touch™ Real-Time PCR
Detection System (catalog #1855485). Data analysis wasperformed using Precision Melt Analysis Software(catalog #1845015).
Protein Expression Analysis

The V3 Western Workflow was used to determine protein
expression. Briefly, 25 µg of cell lysate was applied
to 12% Mini-PROTEAN® TGX™ Gels (catalog #4561043). After
protein separation and blotting, the presence or absence ofBUB1B protein was confirmed using PrecisionAb™ Validated
Western Blotting Antibodies directed against BUB1B(monoclonal antibody, catalog #vma00256). Actin wasvisualized with a rhodamine-conjugated anti-actin human Fabfragment antibody (catalog #12004163). Chemiluminescence
detection was performed using Clarity™ Western ECL
Substrate (catalog #1705061). Western blot image analysis
was performed using the ChemiDoc™ MP Imaging System(catalog #17001402).
Gene Expression Analysis

The PrimePCR™ Cell Cycle Tier 1–4 Panel with 379 genes and5 controls was used to quantify changes in gene expression
between the edited and wild-type eHAP-1 cell lines. cDNA wasprepared from control wild-type and BUB1B-edited eHAP1
RNAs using iScript™ gDNA Clear cDNA Synthesis Kit (catalog#1725034). SsoAdvanced™ Universal SYBR® Green Supermix(catalog #1725270) was used to analyze 10 ng cDNA perreaction on a total of six PrimePCR Cell Cycle Tier 1–4 Panelplates with samples run in triplicate. Plates were run using one
CFX Automation System II (catalog #1845075, robotic platehandler) connected to two CFX384 Touch™ Real-Time PCR
Detection Systems (catalog #1855485). Data were analyzed
using CFX Maestro Software v1.1 (catalog #12004110).

© 2018 Bio-Rad Laboratories, Inc.

Results and Discussion
Our Haploid Gene Editing Workflow provides a fast andefficient method for the generation, analysis, and confirmationof edits for downstream cell-based assays.
Population-Level Screening

Droplet Digital PCR (ddPCR) NHEJ drop-off assays allowed us
to identify and quantify the presence of edits and determine
the percentage of edited cells using population-levelscreening. This is an alternative to incorporating markers,
such as GFP, into editing strategies and enriching for edited
cells by selecting only those cells that show fluorescence. The
rapid readout enabled by ddPCR technology allows quick
optimization of transfection assay conditions by adjusting theamount of cells used and switching from electroporation tolipid-based transfection methods to achieve the most efficienttransfection workflow.
Using the NHEJ drop-off assay, we were able to determinethat after initial transfection none of our clones contained
100% edited cells; data shown for one of our clones, BUB1B-1,
demonstrates 25% edited cells (Figure 2A). To achieve a clonal
population, single cells from edited clones were isolated andfurther expanded. The initial fractional abundance of edits
was used to identify how many single cells to pick and carryforward for each clone. For example, for BUB1B-1 with 25%
edited cells, six clones were picked to ensure at least one
clone that contained the desired edits was carried forward.
This reduces the number of clones to carry forward, thereby
reducing costs of plastics and media.
A
10,000
9,000
8,000
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0

Channel 1 AmplitudeGene Editing

0

1,000

2,000

3,000

4,000

5,000

6,000

Channel 2 Amplitude

B
10,000
9,000
8,000
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0

Channel 1 Amplitude

Materials and Methods

0500

1,000
1,500
Channel 2 Amplitude

2,000

2,500

Fig 2. 2-D plots of NHEJ ddPCR drop-off assay results. Droplets that
contain unedited DNA are positive for both FAM and HEX signal (shown in red).
Droplets that contain NHEJ-edited DNA are positive for FAM only (shown inblue). A, edited DNA from a nonclonal population of cells (clone BUB1B-1) andB, edited DNA from a clonal population of edited cells (clone BUB1B-2).

Bulletin 7079

Fast and Efficient Workflow to Monitor CRISPR-Cas9 Gene Editing of Haploid Cell Lines

After isolation and enrichment, the NHEJ assay wasperformed again, and clone BUB1B-2 was found to be a fully
clonal population with 100% edited cells (Figure 2B).
Clonal Screening

Further confirmation of edits was performed at the clonal level
using HRM analysis. HRM provides orthogonal data to confirm
differences in genetic sequences as a result of gene editing.
We used HRM in place of mismatch assays, which are morelaborious to perform and not as sensitive. The HRM curves
generated from edited and wild-type BUB1B clones showedsignificant differences (Figure 3). These differences suggest
changes in target sequences and are indicative of successful
gene editing.

Protein Expression Confirmation

In the event of an in-frame edit instead of complete removal ofa gene, some protein expression may still occur. Western blot
analysis is a powerful tool for assessing the extent of protein
expression knockdown in edited cells before the cell line isused in downstream assays. We were able to streamline thisprocess by using the V3 Western Workflow, which quantifies
protein expression levels in hours rather than days. We usedPrecisionAb Western Blotting Antibodies, which have beenvalidated, thereby reducing the need to perform additional
assays to confirm their activity and specificity; however, ifrequired, control lysates are provided as positive controls forsuch assays. We saw no expression of the BUB1B protein inthe edited BUB1B-2 clone (Figure 5).
1230.06

Difference

0.04
0.02

BUB1B

0.00
-0.02
Actin-0.04

77.5		

78.0		
78.5		
Temperature, ºC

79.0

Fig. 3. HRM analysis of single-cell clones. HRM analysis of single-cellclones harvested in SingleShot Lysis Buffer without DNase. Precision Melt
Analysis Software was used to plot the melt curves. Wild-type clone (—);
BUB1B-2 clone (—).

Genetic Sequence Analysis

Sanger sequencing results (Figure 4) validated the NHEJ andHRM assay results by providing the sequence of the editedclones. In this example, the edited BUB1B-2 clone had a5 bp deletion in exon 3, which inserted a stop codon after
231 nucleotides of the mRNA.
Wild type
68

69

70

71

72

73

74

75

76

77

78

79

80

TTT

TAC

ACT

GGA

AAT

GAC

CCT

CTG

GAT

GTT

TGG

GAT

AGG

Phe

Tyr

Thr

Gly

Asn

Asp

Pro

Leu

Glu

Val

Trp

Asp

Arg

BUB1B-2
68

69

70

71

72

73

74

75

76

77

78

TTT

TAC

ACT

GGA

AAC

TCT

GGA

TGT

TTG

GGA

TAG

Phe

Tyr

Thr

Gly

Asn

Ser

Gly

Cys

Leu

Gly

*7980

Fig. 4. Sanger sequencing data for BUB1B-2 clone. Sequencing datashow a 5 bp deletion that results in a frameshift with a premature stop codon.
Deleted 5 bp are shown in blue. Changes in the BUB1B-2 nucleotide andprotein sequence, including the early stop codon (*) that results from thisframeshift mutation, are shown in red.

© 2018 Bio-Rad Laboratories, Inc.

Fig. 5. Western blot analysis of wild-type and knockout BUB1B-2 cells.
Lane 1, Precision Plus Protein™ All Blue Prestained Protein Standards(catalog #1610373); lane 2, wild-type BUB1B clone; lane 3, BUB1B knockout
clone BUB1B-2; wild-type BUB1B, 120 kD; actin loading control, ~42 kD.

Gene Expression Analysis

Many assays can be run downstream of edited cell line
production. In knockout studies it is common to explore gene
expression profiles not only of the affected gene(s) but also of
related genes or pathways to look for interdependencies.
By using two CFX Real-Time PCR Detection Systems connectedto the CFX Automation System II we were able to collect data fromtwelve 384-well plates and measure the expression of genes fromthree different screening panels in under 4.5 hours. We comparedgene expression of the wild type with the BUB1B knockout
across 379 genes using the PrimePCR Cell Cycle Tier 1–4 Panel(Figure 6). Analysis using CFX Maestro Software identified fivegenes that were upregulated in the BUB1B knockout: TP63,CRYAB, PRKCH, THBS1, and AC011257.1. Additionally, five

Bulletin 7079

Fast and efficient workflow to monitor CRISPR-Cas9 gene editing of haploid cell lines

genes were found to be downregulated: TP73, UBE2S, GAS6,YEATS4, and, as expected, BUB1B. Interestingly, PRKCH,
a gene encoding a serine- and threonine-specific protein
kinase similar to BUB1B, is upregulated in the knockout cellline, suggesting that the upregulation of this kinase may be
upregulated to compensate for loss of BUB1B function.
272523BUB1B normalized expression

2-1
2-3
AC011357.1

BUB1B

2-7

UBE2S

THBS1
PRKCH
CRYAB

2-9
2-11

TP73
GAS6

YEATS4

2-13
2-15

TP63

2-17
2-19
2-20

2-17

2-14

2-11

2-8

2-5

2-2

2124

27

eHAP1 normalized expression

Fig. 6. Gene expression analysis of BUB1B-2 knockout cells. A scatter plot
provides a view of changes in expression. The red line represents a fourfold
increase and the green line a fourfold decrease in expression compared tocontrol cells (eHAP-1 wild type).

Conclusion
We have demonstrated a simplified workflow that generated a
BUB1B human haploid knockout cell line in less than five weeks.
We were able to obtain robust data in a compressed timeline bymaking the following adjustments to traditional workflows:
■■

■■

■■

■■

■■

Utilizing validated antibodies that eliminate additional assay
time and cost by removing the need to perform in-houseconfirmation of antibody activity and specificity
Pairing the CFX Real-Time PCR Detection System with theCFX Automation System II to reduce hands-on timeTaking advantage of CFX Maestro Software to allow easy
identification of changes in gene expression in response toedits to the genome

Edited cell lines are key to many stages of drug development,from basic research to understanding the key pathways
and associations between genes and/or proteins implicatedin disease to downstream assays where the efficacy ofpotential drugs is being measured in cell-based models.
The ability to generate edited cell lines within a shorter timeframe using our Haploid Gene Editing Workflow can help
accelerate these processes and support faster drug discoveryand development.

212-5

■■

Using Droplet Digital PCR NEHJ drop-off assays to provide
a quick readout of the fractional abundance of edited cells
instead of using reporter genes/markers
Replacing more laborious, less sensitive mismatch assays
with HRM analysis for confirmation of changes togenetic sequences
 mploying the V3 Western Workflow for protein quantitation
Eto save up to a day in assay time and to provide confirmatory
data at each stage of the western blotting workflow

Acknowledgements
We acknowledge the critical work of Meiye Wu, PhD; StevenOkino, PhD; Gerald Uy; Deanna Woo; Mark Shulewitz, PhD;
and Yan Wang, PhD from Reagent R&D, LSG, Bio-Rad
Laboratories, Inc. for the generation of the haploid cell lines.
Visit bio-rad.com/web/GeneEditingHaploid for more
information.
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Patent Nos. 9,089,844; 9,126,160; 9,216,392; 9,347,059; 9,500,664; 9,636,682;
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