Cell lines are the core of biological research. Scientists need cell lines for drug development, basic biology research, safety testing, and biologic therapeutic production. Since the 1980s, genetic manipulation has allowed researchers to tailor cell lines to the experiment or production purpose. Over time, the requirements for these cell lies have risen. In many cases, the cells require multiple genetic edits and must produce data that passes FDA. Moreover, the current funding environment often requires rapid delivery of these cells so scientists can produce data to support further budget and/or investment. This is particularly acute for knock-in cell lines. Current technologies may take months to complete a cell line, allow a limited number of edits, and often have off-target effects that are not suitable for FDA filings. ExpressCells uses its patented FAST-HDR plasmid--along with CRISPR, to address these problems. The FAST-HDR process can precisely knock-in multiple genes (while supporting other types of genetic modifications), ensure precise placement of these edits, and deliver them months faster than competing technologies. This webinar will discuss the basis of the FAST-HDR technology and illustrate several uses. The first part is a presentation by Oscar Perez-Leal, MD, the inventor of the technology. Oscar will discuss the problems he faced as a researcher and how FAST-HDR was designed to address them. He will outline the details of the technology, the history of its development, and several examples where he used FAST-HDR. The second part is a conversation with Jon Weidanz, PhD. Jon will outline the challenges he faced at AbeXXa and how he selected a FAST-HDR custom cell line for his project. He'll outline the learnings from using this cell line, some of which were unexpected, but valuable to future development. By attending this program, attendees will: - Understand the current challenges in creating custom gene-edited cell lines - Know the technology underlying the FAST-HDR gene-editing system, including its use with CRISPR - Be able to describe the advantages of the FAST-HDR system - Learn about several case studies using gene-edited cell lines

1. Welcome!
Creating Better
Gene-Edited Cell Lines
with the FAST-HDR System
Jon Weidanz, MPH, PhD
North Texas Genome Center
University of Texas at Arlington
Founding Director
Oscar Perez-Leal, MD
Assistant Professor
Pharmaceutical Sciences Department
Temple University School of Pharmacy

2. Better knock-ins, better cell lines, better science.

3. Types of Projects
• Core technology: knock-in cell lines built through antibiotic
selection and proprietary plasmid system
• C-terminal tagging
• Overexpressing cell lines
• Point mutations—knock-out target portion of gene and replace with
no sequence
• Can also produce knock-outs using standard CRISPR techniques

4. Phase 1: Design Work
The process starts by analyzing the customer request
to ensure it is feasible. Factors that we analyze are:
• Size of the genetic insertion
• Location in the gene
• Is the gene expressed by the particular cell line?
• Is the gene essential?
If the project is feasible, a molecular biologist will
design both a CRISPR and the insertion plasmid, which
will include the targeted insertion, an antibiotic
selection gene, and often, a gene coding for a tag.
3

5. Phase 1: Design Work Phase 2: Bacteria Work
ExpressCells uses E. coli to produce the CRISPR and
insertion cassettes
4

6. Phase 1: Design Work Phase 2: Bacteria Work
CRISPR
Antibiotic
Cell Pool (Polyclonal)
Single Cell Dilution
Phase 3: Cell Culture
Homozygous
Heterozygous
Heterozygous
We then culture the cells. This
includes:
• Insertion of the plasmids into the
cell (via electroporation or
transfection)
• Introduction of the antibiotic to
remove cells without the genetic
change to create a cell pool
• Single cell dilution to identify
clones

7. Phase 1: Design Work Phase 2: Bacteria Work
CRISPR
Antibiotic
Cell Pool (Polyclonal)
Single Cell Dilution
Homozygous Heterozygous
Monoclonal
?
?
Phase 3: Cell Culture
Homozygous
Heterozygous
Heterozygous

8. C-tagging Knock-in Cell Lines in < 14 Days
Create
donor
vector: 1 d
Isolate pure, modified cells: ≈ 10 d
FAST-HDR
Create
donor
vector: 21 d
Isolate pure, modified cells: 4 – 8 weeks
Conventional methods

9. FAST-HDR Plasmid Vector System
Tag P2A Res. gene
Left recombination arm Right recombination arm
Left recombination arm Right recombination arm
Synthetic or PCR-generated DNA Synthetic or PCR-generated DNA
ccdB
Tag P2A Res. gene
ccdB
Directional
cloning site
Directional
cloning site
+
donor
HDR
vector
Tag marker P2A Resistance gene
Puromycin
6-
H
I
S
mClover 3
6-
H
I
S
mRuby3
6-
H
I
S
mTagBFP2
6-
H
I
S
Luciferase
Zeocin™
Blastocidin
C
B
A
D

10. ExpressCells
Live cells Cell microscopy
Direct image analysis
(0 min)
Traditional Screening
Live cells Additional staining
(20 – 60 min)
1st Ab
(1 – 16 h)
Cell fixing
Cell permeabilization
Ag blocking
(60 – 90 min)
Cell microscopy
2nd Ab
(1 – 6 h)
Triple-Labeling With FAST-HDR Obviates the
Need for Immunofluorescence and Staining

11. Endogenous, Single Gene Labeling
Tagging With mRuby3, Selected With Zeocin™,
Day 14 Following Transfection

12. Double Gene Labeling
Tagging With mRuby3 + mClover,
Selection With Zeocin™ + Puromycin

13. Triple Gene Labeling
ATP5B Tagging With mTagBFP2, β-tubulin With mClover3,
Histone H3.3 With mRuby3

14. Flexible Approach
Mitochondria
Red: H3.3-mRuby3
Green: β-tubulin
Spindle poles
Red: H3.3-mRuby3
Green: β-tubulin
Nucleus
Red: H3.3-mRuby3
Green: β-tubulin

15. Two Case Studies
Problem: Does a New
Oncology Agent Inhibit Mitosis?
Solution: Create cell line where cell
division is readily identified via blue
tag for protein expressed only
during mitosis
Problem: Identify Impact of a
Drug Candidate on Organelles
14
Solution: Insert three genes that
clearly identify the cytoskeleton,
nucleus, and mitochondria

16. Better High-Content Imaging With the
FAST-HDR Plasmid Vector System
• Longitudinal compound library screening
• Better target- or phenotype-based hit identification
• Longitudinal cell-based toxicology assays
• Longitudinal study of inhibitors of intracellular signaling pathways
• Discrimination among protein sequence variants
• Defining protein–membrane and protein–protein interactions without
fixation and staining
• Rapid homozygous tagging of target genes

17. Better knock-ins, better cell lines, better science.
xpresscells.com info@xpresscells.com +1 (484) 483-6759

18. Thank you for
participating!