In this study, we developed a CRISPR/Cas9-based high throughput loss-of-function screen for identifying target genes responsible for the tumor proliferation and growth in TNBC. Our initial focus was to identify essential kinases in MDA-MB-231 cell line using the Invitrogen™ LentiArray™ Human Kinase CRISPR Library, which targets 840 kinases with up to 4 different gRNAs per protein kinase for complete gene knockout. This functional screen identified over 90 protein kinases that are essential for cell viability and cell proliferation. Ten of these hits (CDK1, CDK2, CDK8, CDK10, CDK11A, CDK19, CDK19, CDC7, EPHA2 and WEE1) are well-known targets validated in the literature. Currently, we are in the process validating the novel hits through target gene sequencing, western blotting and target specific small molecule kinase inhibitors.

1. Tufan Gokirmak1, Chetana Revankar1, Jason Sharp1, David R. Piper1; Thermo Fisher Scientific, Carlsbad (1), USA
ABSTRACT
Breast cancer is the most commonly diagnosed cancer among women in worldwide and the second
leading cause of death by cancer after lung cancer. Breast cancer can be broadly grouped into three main
groups: HR+ (estrogen receptor positive and/or progesterone receptor positive), HER2+ (human
epidermal growth factor receptor 2 positive) and triple-negative (HR- and HER2-). Hormone and anti-
HER2 targeted therapies are available for receptor positive breast cancer types. However, since triple-
negative breast cancer (TNBC) does not have estrogen, progesterone or HER2 receptors, there are no
such targeted therapies available for this type of cancer. Currently, oncologists are resort to treating TNBC
patients with traditional chemotherapy drugs, surgery and radiation. Thus, due its limited treatment options
and poor prognosis, search for specific molecular targets and developing novel treatments for TNBC is
one of the highest priorities of current breast cancer research.
In this study, we developed a CRISPR/Cas9-based high throughput loss-of-function screen for identifying
target genes responsible for the tumor proliferation and growth in TNBC. Our initial focus was to identify
essential kinases in MDA-MB-231 cell line using the Invitrogen™ LentiArray™ Human Kinase CRISPR
Library, which targets 840 kinases with up to 4 different gRNAs per protein kinase for complete gene
knockout. This functional screen identified over 90 protein kinases that are essential for cell viability and
cell proliferation. Ten of these hits (CDK1, CDK2, CDK8, CDK10, CDK11A, CDK19, CDK19, CDC7,
EPHA2 and WEE1) are well-known targets validated in the literature. Currently, we are in the process
validating the novel hits through target gene sequencing, western blotting and target specific small
molecule kinase inhibitors.
INTRODUCTION
Target identification and validation leading to novel and druggable targets for therapeutic
intervention remains a top priority for the pharmaceutical and biotechnology industry when it comes to
building a robust drug discovery pipeline. However, there are still significant challenges to translate the
potential targets to clinical trial outcomes. Therefore, functional genomic approaches may provide the
next-generation platforms to effectively bridge these gaps.
In this work, we focused on protein kinases, since they are involved in cell signaling cascades leading to
cell proliferation, migration and survival in all cell types, and their dysregulation often leads to cancer. Due
to these oncogenic roles, they have become one of the most important classes of drug targets in the
field of cancer biology. Therefore development of novel high-throughput functional screening platforms
that enable the identification of protein kinases or for that matter any gene, essential for tumor proliferation
and viability is expected to advance oncogenic drug discovery efforts.
CRISP/Cas9 system is a versatile genome editing technology that enables gene specific knockouts for
early target discovery (Figure 1). In this study, we utilized CRISPR/Cas9 system to target 840 kinases
individually and discovered their roles in triple-negative breast cancer cell viability and proliferation.
CONCLUSIONS AND FUTURE DIRECTIONS
Development of novel high-throughput screening platforms that enable identification of genes that are essential for
tumor proliferation and viability is expected to advance the cancer research and oncogenic drug discovery efforts. In
this study, we established a clonal MDA-MB-231-Cas9 cell line that stably expresses Cas9 protein, which allows
targeted and efficient gene knockouts to study the roles of any gene of interest in cell proliferation and viability. Next,
we optimized assay parameters to run high-throughput functional screenings for cancer cell viability and proliferation
using the Invitrogen™ LentiArray™ Human Kinase CRISPR Library. Our screening results showed that the 10 of the
top 10% of the hits detected by the PrestoBlue™ Cell Viability and CyQUANT™ Direct Cell Proliferation are known
protein kinases involved in cell cycle regulation. In addition, we discovered over 80 novel targets that may be
essential for cell viability and proliferation in TNBC. Our current efforts focus on the validation of these hits by next
generation sequencing, gene expression, and western blot analyses to show that the gene sequences that encode
the identified hits are disrupted by the Cas9 and the protein encoded by the identified target genes are depleted. Our
next goal is to mimic these functional assays with known small molecule kinase inhibitors and eventually run screens
with small molecule libraries against validated targets to identify lead compounds that can interfere with TNBC cell
viability and proliferation.
TRADEMARKS
© 2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific
and its subsidiaries unless otherwise specified. For Research Use Only. Not for use in diagnostic procedures.
Identifying novel and druggable targets in a triple negative breast cancer cell line using the Invitrogen™ LentiArray™
Human Kinase CRISPR Library
Thermo Fisher Scientific • 5781 Van Allen Way • Carlsbad, CA 92008 • www.thermofisher.com
Figure 3. Isolation and validation of MDA-MB231-Cas9 clones. Single cells were sorted into 96-well plates
and clonally expanded via FACS. Genomic cleavage efficiency at HPRT locus was determined for each clone
using the GeneArt® Genomic Cleavage Detection Assay. Clone 4 and 5 have the highest cleavage efficiency.
Comparison of parental MDA-MB-231 with MDA-MB231-Cas9 clones indicated that cell proliferation of clone 5 is
identical to the parental MDA-MB-231 cells.
Figure 4. Functional genomics assays in MDA-MB-231 cells using the Invitrogen™ LentiArray™ Human
Kinase CRISPR Library. The functional screens identified over 90 hits that interfere with cell viability and cell
proliferation. 71 of the hits were detected by both PrestoBlue™ Cell Viability and CyQUANT™ Direct Cell
Proliferation assays. Each assay also detected 13 unique targets that are involved specifically in cell viability or cell
proliferation.
Blasticidin selection for
3 weeks
MDA-MB-231
PrestoBlue™ Cell Viability
CyQUANT™ Direct
Cell Proliferation Assay
Top%10hitsTop%10hits
Assay Optimization Summary
Cell Seeding: 2000 cells/well
MOI: 4
Puromycin selection: √
Spinoculation: √
Positive controls: CHEK1 and PLK1
Negative control: Scramble
2- Assay optimization
3- LentiArray™ CRISPR Human Kinase Screen
1- Stable Cas9-expressing cell line generation RESULTS
GeneArt™ Genomic
Cleavage Detection Assay
Indel (%): 89LentiArray™ CRISPR
Positive Control Lentivirus,
human HPRT, with GFP
Clonal isolation and validation
Invitrogen™ LentiArray™
Cas9 Lentivirus
Lentiviral vector construction
Invitrogen™ LentiArray™
Cas9 Lentivirus
Invitrogen™ LentiArray™
Lentiviral gRNA
MDA-MB-231-Cas9 pool validation
Figure 1. CRISPR/Cas9 for gene kockout. A CRISPR-Cas9 targeted double-strand break. Cleavage occurs on
both strands, 3 bp upstream of the NGG in the protospacer adjacent motif (PAM) sequence on the 3´ end of the
target sequence.
Figure 2. CRISPR/Cas9 lentiviral vector construction. A. Invitrogen™ LentiArray™ Cas9 Lentivirus construct
encodes human codon-optimized S. pyogenes Cas9 protein and blasticidin resistance linked to Cas9 through a
self-cleaving 2A peptide. B. Invitrogen™ LentiArray™ Lentiviral gRNA constructs encode specific sgRNA
transcribed from U6 promoter and puromycin resistance driven by EF-1α promoter.
A
B
High transduction efficiency, ≥90%
67% 73% 62% 84% 87%
CDK1 CDK18
CDK2 CDK19
CDK8 CDC7
CDK10 EPHA2
CDK11A WEE1
Previously validated targets in the literature