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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
Better knock-ins, better cell lines, better science.
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
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
Phase 1: Design Work Phase 2: Bacteria Work
ExpressCells uses E. coli to produce the CRISPR and
insertion cassettes
4
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
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
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
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
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
Endogenous, Single Gene Labeling
Tagging With mRuby3, Selected With Zeocin™,
Day 14 Following Transfection
Double Gene Labeling
Tagging With mRuby3 + mClover,
Selection With Zeocin™ + Puromycin
Triple Gene Labeling
ATP5B Tagging With mTagBFP2, β-tubulin With mClover3,
Histone H3.3 With mRuby3
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
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
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
Better knock-ins, better cell lines, better science.
xpresscells.com info@xpresscells.com +1 (484) 483-6759
Thank you for
participating!

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Creating Better Gene-Edited Cell Lines with the FAST-HDR System

  • 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