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©2015 BioInformatics LLC www.gene2drug.com 1
BioInformatics LLC
2111 Wilson Boulevard
Suite 250
Arlington, VA 22201
703.778.3080
703.778.3081 fax
www.gene2drug.com
The CRISPR/Cas9 Toolbox
August 2015
Get access to The Market for CRISPR/Cas9 Genome Editing
Products, on the Interactive Market Intelligence Platform
©2015 BioInformatics LLC www.gene2drug.com 2
Overview
 Following the insights gained from BioInformatics’ report, The Market for
CRISPR/Cas9 Genome Editing Products, released in June 2015 on the
Interactive Market Intelligence platform, a follow-up questionnaire was
sent to The Science Advisory Board to quantify how the emergence of
CRISPR/Cas9 technology is impacting the use of traditional tools and
techniques. It was fielded between July 6-8, 2015.
 We are sharing the results, arranged in three parts:
 Part I: The Market for CRISPR/Cas9 Genome Editing Products on Interactive
Market Intelligence
 Part II: CRISPR’s impact on current tools/techniques
 Part III: Verbatim responses from panelists on how to improve the CRISPR
workflow and validation process
©2015 BioInformatics LLC www.gene2drug.com 2
©2015 BioInformatics LLC www.gene2drug.com 3
PART I
The Market for CRISPR/Cas9 Genome Editing
Products on Interactive Market Intelligence
©2015 BioInformatics LLC www.gene2drug.com 4
About Interactive Market Intelligence (IMI)
 The Market for CRISPR/Cas9 Genome Editing Products report on the
Interactive Market Intelligence platform will help you:
 Estimate market size, share and growth by region, industry segment and
product category
 Understand the drivers of brand loyalty and compare brand performance
 Measure customer satisfaction with CRISPR/Cas9 products
 Highlight the strengths and weaknesses of competitors and their customers’
likelihood-to-switch
 For more information and to subscribe to the IMI report, visit:
 Web: www.gene2drug.com/CRISPR
 Download the report brochure.
 Contact Zach French at z.french@gene2drug.com or at 703-778-3080
©2015 BioInformatics LLC www.gene2drug.com 4
©2015 BioInformatics LLC www.gene2drug.com 5
PART II
CRISPR’s impact on current tools/techniques
©2015 BioInformatics LLC www.gene2drug.com 6
Part I: CRISPR’s impact on current tools/techniques
©2015 BioInformatics LLC www.gene2drug.com 6
The CRISPR/Cas9 Workflow
Identify
Target
Sequence
Prepare
CRISPR
Construct
Introduce
CRISPR/Cas9
to Competent
Cells
Identify
Successfully
Altered Cells
Confirm
Mutations
©2015 BioInformatics LLC www.gene2drug.com 7
Part I: CRISPR’s impact on current tools/techniques
©2015 BioInformatics LLC www.gene2drug.com 7
Which of the following techniques do you use to confirm your CRISPR construct? (Check all the
apply)
n=316
53%
51%
46%
42%
22%
6%
2%
End point PCR
Sanger sequencing
Gel electrophoresis
Real-Time PCR
Next Generation Sequencing
Digital PCR
Other
©2015 BioInformatics LLC www.gene2drug.com 8
Part I: CRISPR’s impact on current tools/techniques (cont’d)
©2015 BioInformatics LLC www.gene2drug.com 8
How do you expect your use of the following techniques used to confirm your CRISPR construct
to change as a result of your use of CRISPR/Cas9 technology? (Choose only one for each)
20%
15%
33%
26%
30%
27%
11%
14%
22%
17%
9%
5%
9%
10%
15%
4%
4%
7%
4%
4%
20%
49%
49%
23%
32%
30%
49%
8%
10%
5%
6%
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
End point PCR
(n=169)
Gel electrophoresis
(n=145)
Next Generation Sequencing
(n=69)
Real-Time PCR
(n=133)
Digital PCR
(n=20*)
Sanger sequencing
(n=161)
Increase by >5% Increase by 5% Increase by 4% Increase by 3%
Increase by 2% Increase by 1% Stay the same Decrease
©2015 BioInformatics LLC www.gene2drug.com 9
Part I: CRISPR’s impact on current tools/techniques (cont’d)
©2015 BioInformatics LLC www.gene2drug.com 9
Which transfection method(s) do you use to introduce CRISPR/Cas9 constructs into target cells?
(Check all that apply)
57%
56%
39%
4%
Reagent-based (such as
lipofection, DEAE-dextran, etc.)
Instrument-based (such as
electroporation, microinjection)
Viral vectors
Other
n=316
©2015 BioInformatics LLC www.gene2drug.com 10
Part I: CRISPR’s impact on current tools/techniques (cont’d)
©2015 BioInformatics LLC www.gene2drug.com 10
How do you expect your use of the following methods used to introduce CRISPR/Cas9 constructs
into target cells to change as a result of your use of CRISPR technology? (Choose only one for
each)
26%
22%
34%
17%
8%
13%
6%
10%
6%
6%
5%
35%
42%
35%
9%
7%
5%
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Instrument-based (e.g.
electroporation, microinjection)
(n=176)
Reagent-based (e.g. lipofection,
DEAE-dextran, etc.)
(n=180)
Viral vectors
(n=124)
Increase by >5% Increase by 5% Increase by 4% Increase by 3%
Increase by 2% Increase by 1% Stay the same Decrease
©2015 BioInformatics LLC www.gene2drug.com 11
Part I: CRISPR’s impact on current tools/techniques (cont’d)
©2015 BioInformatics LLC www.gene2drug.com 11
Which of the following techniques do you expect to use to confirm you have successfully altered
the target cells? (Check all that apply)
n=316
62%
49%
44%
36%
21%
13%
9%
8%
7%
Real-Time PCR
Fluorescent microscopy
Function-specific cell based assay
Flow cytometry and/or FACS
ELISA assay
High Content Screening
Digital PCR
Other
Label Free Detection
©2015 BioInformatics LLC www.gene2drug.com 12
23%
20%
17%
15%
23%
5%
25%
37%
9%
14%
11%
20%
14%
10%
13%
10%
4%
6%
12%
10%
10%
5%
8%
9%
5%
4%
29%
8%
7%
5%
6%
5%
14%
4%
9%
4%
4%
5%
49%
35%
43%
37%
41%
29%
38%
27%
5%
6%
7%
7%
7%
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Fluorescent microscopy
(n=155)
ELISA assay (n=65)
Flow cytometry and/or FACS
(n=115)
High Content Screening (n=41)
Function-specific cell based
assay (n=138)
Label Free Detection (n=21*)
Real-Time PCR (n=197)
Digital PCR (n=30)
Increase by >5% Increase by 5% Increase by 4% Increase by 3%
Increase by 2% Increase by 1% Stay the same Decrease
Part I: CRISPR’s impact on current tools/techniques (cont’d)
©2015 BioInformatics LLC www.gene2drug.com 12
How do you expect your use of the following techniques used to successfully alter target cells to
change as a result of your use of CRISPR/Cas9 technology? (Choose only one for each)
©2015 BioInformatics LLC www.gene2drug.com 13
Part I: CRISPR’s impact on current tools/techniques (cont’d)
©2015 BioInformatics LLC www.gene2drug.com 13
Which of the following techniques do you use to confirm mutations in DNA? (Check all that
apply)
n=316
61%
43%
37%
30%
27%
12%
6%
6%
1%
Sanger sequencing
Endpoint PCR
Real-Time PCR
Western blot
Next Generation Sequencing
Fluorescent microscopy
Microarray
Digital PCR
Other
©2015 BioInformatics LLC www.gene2drug.com 14
Part I: CRISPR’s impact on current tools/techniques (cont’d)
©2015 BioInformatics LLC www.gene2drug.com 14
How do you expect your use of the following techniques used to confirm mutations in DNA to
change as a result of your use of CRISPR/Cas9 technology? (Choose only one for each)
12%
21%
10%
33%
20%
30%
20%
20%
8%
10%
25%
13%
10%
10%
9%
6%
5%
13%
10%
7%
6%
5%
7%
5%
10%
7%
7%
10%
5%
6%
7%
5%
7%
9%
5%
4%
10%
4%
5%
4%
7%
10%
5%
50%
41%
40%
26%
36%
30%
49%
48%
7%
5%
5%
5%
5%
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Endpoint PCR (n=135)
Fluorescent microscopy (n=39)
Microarray (n=20*)
Next Generation Sequencing
(n=84)
Real-Time PCR (n=117)
Digital PCR (n=20*)
Sanger sequencing (n=193)
Western blot (n=94)
Increase by >5% Increase by 5% Increase by 4% Increase by 3%
Increase by 2% Increase by 1% Stay the same Decrease
©2015 BioInformatics LLC www.gene2drug.com 15
PART II
How to improve the CRISPR workflow and validation
process (Verbatim responses)
©2015 BioInformatics LLC www.gene2drug.com 16©2015 BioInformatics LLC www.gene2drug.com 16
Question:
A majority of scientists said ‘validation of
genome editing results’ was the one area they
would most like to improve.
How would they propose solving this problem?
©2015 BioInformatics LLC www.gene2drug.com 17
Question: A majority of scientists said ‘validation of genome editing results’
was the one area they would most like to improve. How would you propose
solving this problem?
©2015 BioInformatics LLC www.gene2drug.com 17
Staff Scientist, Singapore:
“Ensure the targeting is unique and specific. Southern blots to confirm that only specific targeting
events have occurred using probes is required to ensure there are no other insertions. Otherwise,
DNA-Seq to check whole genome is a reliable albeit expensive validation method.”
Senior Scientist, United Kingdom:
“Good quality antibody reagents and good functional assays. Also,
using other techniques such as site-directed mutagenesis and RNAi
knockdown to understand the biology more fully.”
Senior Scientist, United States:
“The best way we have found to validate genome editing is to move to an in vivo system by
SCNT and cloning. Further analysis and characterization of the genotype and phenotype of
the resulting animals provide us with the most complete data sets.”
©2015 BioInformatics LLC www.gene2drug.com 18©2015 BioInformatics LLC www.gene2drug.com 18
Postdoctoral Fellow, United States:
“Sequencing to detect the point of insertion, similar to DNA modifications, enrich for guide RNA
insertions with a probe and sequence fragments that are bound to the probe.”
Postdoctoral Fellow, Australia:
“I don’t see any obvious possible improvement. I think the design of the target should be the main
focus to avoid off target effects which are much more difficult to identify.”
Laboratory Technician, United States:
“A possibility is to make results "open source" so that data can be shared amongst scientists that
are aiming to modify a specific gene. If they're experiencing similar results (seq data, behavior,
etc.) then we may be more confident that the editing results are trustworthy. This could still be
problematic as everyone might have similar off-target effects then we'd need to consider using
bioinformatics to identify sequence homologies that may result in these off-target sites and seq
those regions. Whole-genome seq might be another avenue to validate but that option is costly. ”
Question: A majority of scientists said ‘validation of genome editing results’
was the one area they would most like to improve. How would you propose
solving this problem?
©2015 BioInformatics LLC www.gene2drug.com 19©2015 BioInformatics LLC www.gene2drug.com 19
PhD Student, Germany:
“Next gen amplicon seq would be the fastest and most accurate way to screen for modified clones.
Applications which use nickase Cas9 or delivery of complex Cas9-gRNA could also minimize off
targets while targeting the site of interest. According though to the application of editing, different
enrichment methods could be used such as FACS for sorting out tagged proteins.”
Bioengineer, France:
“To ensure the validation of genome editing results, we use target specific real-time PCR or in
some cases we select edited populations under selective pressure.”
Laboratory Director, United States:
“The only way to be sure is to do a functional protein assay or western blot, but this is impossible
for all the proteins that may be altered, so using a control consisting of a bacterial or yeast gene
that means nothing to the cell and showing that both cells behave the same except for the
mutation.”
Question: A majority of scientists said ‘validation of genome editing results’
was the one area they would most like to improve. How would you propose
solving this problem?
©2015 BioInformatics LLC www.gene2drug.com 20©2015 BioInformatics LLC www.gene2drug.com 20
Professor, China:
“Using next generation sequencing technology and PCR with specific primers.”
Professor, Canada:
“Next generation sequencing probably is the best method at the moment.”
Principal Investigator, United States:
“It would be helpful to use a genome capture and deep sequencing approach to identify edits in
clones. However, this is expensive and needs to be refined to allow for a more cost effective and
practical use.”
Postdoctoral Fellow, Norway:
“This can be simplified using specific non-toxic RNA probes.”
Question: A majority of scientists said ‘validation of genome editing results’
was the one area they would most like to improve. How would you propose
solving this problem?
©2015 BioInformatics LLC www.gene2drug.com 21©2015 BioInformatics LLC www.gene2drug.com 21
PhD Student, India:
“Mutations in targeted genes were analyzed by PCR amplification and Sanger sequencing. Primers
from the vicinity of the targeted regions were used for amplification of control and transformed
samples. Ten cycles of amplification were performed. The amplified DNA was cloned and used for
sequencing. ”
Laboratory Director, United Kingdom:
“You have to use a range of techniques. Science is testable, mutational research reporter assays
should all give the same result. Gene transcriptome analysis would be the best: comparing gene
changes with a genetic knockdown vs. CRISPR/Cas9 system should give near as identical profiling.”
Staff Scientist, United States:
“The simple way is end point PCR as a first diagnostic step followed by phenotypic changes.”
Question: A majority of scientists said ‘validation of genome editing results’
was the one area they would most like to improve. How would you propose
solving this problem?
©2015 BioInformatics LLC www.gene2drug.com 22©2015 BioInformatics LLC www.gene2drug.com 22
PhD Student, Philippines:
“There are other ways of validating a results like we do phenotyping of lines that were produced.”
Professor, Italy:
“On-target and off-target genome editing should be monitored by next gen sequencing / use of
selection markers could improve the identification of CRISPR/Cas9 edited cells.”
Staff Scientist, United States:
“Will likely remain time consuming, as single colonies need to be picked. Specific PCR, possibly
followed by sequencing, still seems to be the safest method to validate a mutation genetically.”
Postdoctoral Fellow, India:
“I found surveyor nuclease assay to be the best assay for preliminary check. Next we can even go
for NGS to check the indels.”
Question: A majority of scientists said ‘validation of genome editing results’
was the one area they would most like to improve. How would you propose
solving this problem?
©2015 BioInformatics LLC www.gene2drug.com 23©2015 BioInformatics LLC www.gene2drug.com 23
Staff Scientist, United States:
“To validate, confirm the genome editing by sequencing, expression of the target gene by qRT-
PCR/Western blot and finally have to perform functional assay based on the target gene.”
Staff Scientist, United States:
“Depends. For model organisms where off targets are less of a concern, HRMA is easy, sensitive,
and works well. For human cases, deep sequencing is needed.”
Professor, Finland:
“Generation of KIs by inserting a defined short segments such as a series of stop codons in
different frames. ”
Question: A majority of scientists said ‘validation of genome editing results’
was the one area they would most like to improve. How would you propose
solving this problem?
©2015 BioInformatics LLC www.gene2drug.com 24
Demographics (n=316)
©2015 BioInformatics LLC www.gene2drug.com 24
29%
26%
45%
Geographic Region
Asia-Pacific
Europe
North America
80%
20%
Market Segment
Academic
Pharma/Biotech
<1%
1%
1%
2%
2%
4%
8%
11%
16%
17%
17%
19%
QA/QC
Bioengineer
Executive (CEO, VP, etc.)
Laboratory Technician
Other
Department Head
Graduate Student/Research…
Lab…
Post Doctoral Fellow
Professor/Assistant Professor/Teacher
Staff Scientist
Principal Investigator
Job Position
1%
1%
1%
2%
3%
3%
4%
7%
11%
14%
52%
Food/Beverage Manufacturer
Other
Research Institute (for-profit)
Contract Research Organization
Hospital
University Medical Center
Pharmaceutical Company
Government
Biopharmaceutical/Biotechnology Company
Research Institute/Foundation (non-profit)
Academic/University
Employment Sector
©2015 BioInformatics LLC www.gene2drug.com 25
About Us
©2015 BioInformatics LLC www.gene2drug.com 25
BioInformatics LLC, together with our SDi division, is the premier research and
advisory firm serving the life science tools industry. By leveraging our online
professional network of tens of thousands of biomedical researchers, we have
supported more than 400 companies and provided insights that lead to better
business decisions. Our expertise includes assessing the size and attractiveness of
markets, optimizing product configurations and pricing, validating corporate
acquisitions, measuring customers’ brand loyalty, and evaluating brand strength and
positioning.
Since 1994, BioInformatics LLC has been providing off-the-shelf reports, custom-
designed studies and market analysis that enable companies to understand their
market and competitors through the eyes of the most important information source
of all—the people who buy their products.
Zach French
Marketing Manager
703.778.3080 x19
z.french@gene2drug.com
For more information, please contact:
©2015 BioInformatics LLC www.gene2drug.com 26
Interactive Market Intelligence
©2015 BioInformatics LLC www.gene2drug.com 26
Current titles:
The Market for Real-Time PCR
The Market for Cell-Based Assays
The Market for Stem Cell Research Products
The Market for CRISPR/Cas9 Genome Editing Products
For more information, please visit gene2drug.com/IMI.

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The CRISPR/Cas9 Toolbox

  • 1. ©2015 BioInformatics LLC www.gene2drug.com 1 BioInformatics LLC 2111 Wilson Boulevard Suite 250 Arlington, VA 22201 703.778.3080 703.778.3081 fax www.gene2drug.com The CRISPR/Cas9 Toolbox August 2015 Get access to The Market for CRISPR/Cas9 Genome Editing Products, on the Interactive Market Intelligence Platform
  • 2. ©2015 BioInformatics LLC www.gene2drug.com 2 Overview  Following the insights gained from BioInformatics’ report, The Market for CRISPR/Cas9 Genome Editing Products, released in June 2015 on the Interactive Market Intelligence platform, a follow-up questionnaire was sent to The Science Advisory Board to quantify how the emergence of CRISPR/Cas9 technology is impacting the use of traditional tools and techniques. It was fielded between July 6-8, 2015.  We are sharing the results, arranged in three parts:  Part I: The Market for CRISPR/Cas9 Genome Editing Products on Interactive Market Intelligence  Part II: CRISPR’s impact on current tools/techniques  Part III: Verbatim responses from panelists on how to improve the CRISPR workflow and validation process ©2015 BioInformatics LLC www.gene2drug.com 2
  • 3. ©2015 BioInformatics LLC www.gene2drug.com 3 PART I The Market for CRISPR/Cas9 Genome Editing Products on Interactive Market Intelligence
  • 4. ©2015 BioInformatics LLC www.gene2drug.com 4 About Interactive Market Intelligence (IMI)  The Market for CRISPR/Cas9 Genome Editing Products report on the Interactive Market Intelligence platform will help you:  Estimate market size, share and growth by region, industry segment and product category  Understand the drivers of brand loyalty and compare brand performance  Measure customer satisfaction with CRISPR/Cas9 products  Highlight the strengths and weaknesses of competitors and their customers’ likelihood-to-switch  For more information and to subscribe to the IMI report, visit:  Web: www.gene2drug.com/CRISPR  Download the report brochure.  Contact Zach French at z.french@gene2drug.com or at 703-778-3080 ©2015 BioInformatics LLC www.gene2drug.com 4
  • 5. ©2015 BioInformatics LLC www.gene2drug.com 5 PART II CRISPR’s impact on current tools/techniques
  • 6. ©2015 BioInformatics LLC www.gene2drug.com 6 Part I: CRISPR’s impact on current tools/techniques ©2015 BioInformatics LLC www.gene2drug.com 6 The CRISPR/Cas9 Workflow Identify Target Sequence Prepare CRISPR Construct Introduce CRISPR/Cas9 to Competent Cells Identify Successfully Altered Cells Confirm Mutations
  • 7. ©2015 BioInformatics LLC www.gene2drug.com 7 Part I: CRISPR’s impact on current tools/techniques ©2015 BioInformatics LLC www.gene2drug.com 7 Which of the following techniques do you use to confirm your CRISPR construct? (Check all the apply) n=316 53% 51% 46% 42% 22% 6% 2% End point PCR Sanger sequencing Gel electrophoresis Real-Time PCR Next Generation Sequencing Digital PCR Other
  • 8. ©2015 BioInformatics LLC www.gene2drug.com 8 Part I: CRISPR’s impact on current tools/techniques (cont’d) ©2015 BioInformatics LLC www.gene2drug.com 8 How do you expect your use of the following techniques used to confirm your CRISPR construct to change as a result of your use of CRISPR/Cas9 technology? (Choose only one for each) 20% 15% 33% 26% 30% 27% 11% 14% 22% 17% 9% 5% 9% 10% 15% 4% 4% 7% 4% 4% 20% 49% 49% 23% 32% 30% 49% 8% 10% 5% 6% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% End point PCR (n=169) Gel electrophoresis (n=145) Next Generation Sequencing (n=69) Real-Time PCR (n=133) Digital PCR (n=20*) Sanger sequencing (n=161) Increase by >5% Increase by 5% Increase by 4% Increase by 3% Increase by 2% Increase by 1% Stay the same Decrease
  • 9. ©2015 BioInformatics LLC www.gene2drug.com 9 Part I: CRISPR’s impact on current tools/techniques (cont’d) ©2015 BioInformatics LLC www.gene2drug.com 9 Which transfection method(s) do you use to introduce CRISPR/Cas9 constructs into target cells? (Check all that apply) 57% 56% 39% 4% Reagent-based (such as lipofection, DEAE-dextran, etc.) Instrument-based (such as electroporation, microinjection) Viral vectors Other n=316
  • 10. ©2015 BioInformatics LLC www.gene2drug.com 10 Part I: CRISPR’s impact on current tools/techniques (cont’d) ©2015 BioInformatics LLC www.gene2drug.com 10 How do you expect your use of the following methods used to introduce CRISPR/Cas9 constructs into target cells to change as a result of your use of CRISPR technology? (Choose only one for each) 26% 22% 34% 17% 8% 13% 6% 10% 6% 6% 5% 35% 42% 35% 9% 7% 5% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Instrument-based (e.g. electroporation, microinjection) (n=176) Reagent-based (e.g. lipofection, DEAE-dextran, etc.) (n=180) Viral vectors (n=124) Increase by >5% Increase by 5% Increase by 4% Increase by 3% Increase by 2% Increase by 1% Stay the same Decrease
  • 11. ©2015 BioInformatics LLC www.gene2drug.com 11 Part I: CRISPR’s impact on current tools/techniques (cont’d) ©2015 BioInformatics LLC www.gene2drug.com 11 Which of the following techniques do you expect to use to confirm you have successfully altered the target cells? (Check all that apply) n=316 62% 49% 44% 36% 21% 13% 9% 8% 7% Real-Time PCR Fluorescent microscopy Function-specific cell based assay Flow cytometry and/or FACS ELISA assay High Content Screening Digital PCR Other Label Free Detection
  • 12. ©2015 BioInformatics LLC www.gene2drug.com 12 23% 20% 17% 15% 23% 5% 25% 37% 9% 14% 11% 20% 14% 10% 13% 10% 4% 6% 12% 10% 10% 5% 8% 9% 5% 4% 29% 8% 7% 5% 6% 5% 14% 4% 9% 4% 4% 5% 49% 35% 43% 37% 41% 29% 38% 27% 5% 6% 7% 7% 7% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Fluorescent microscopy (n=155) ELISA assay (n=65) Flow cytometry and/or FACS (n=115) High Content Screening (n=41) Function-specific cell based assay (n=138) Label Free Detection (n=21*) Real-Time PCR (n=197) Digital PCR (n=30) Increase by >5% Increase by 5% Increase by 4% Increase by 3% Increase by 2% Increase by 1% Stay the same Decrease Part I: CRISPR’s impact on current tools/techniques (cont’d) ©2015 BioInformatics LLC www.gene2drug.com 12 How do you expect your use of the following techniques used to successfully alter target cells to change as a result of your use of CRISPR/Cas9 technology? (Choose only one for each)
  • 13. ©2015 BioInformatics LLC www.gene2drug.com 13 Part I: CRISPR’s impact on current tools/techniques (cont’d) ©2015 BioInformatics LLC www.gene2drug.com 13 Which of the following techniques do you use to confirm mutations in DNA? (Check all that apply) n=316 61% 43% 37% 30% 27% 12% 6% 6% 1% Sanger sequencing Endpoint PCR Real-Time PCR Western blot Next Generation Sequencing Fluorescent microscopy Microarray Digital PCR Other
  • 14. ©2015 BioInformatics LLC www.gene2drug.com 14 Part I: CRISPR’s impact on current tools/techniques (cont’d) ©2015 BioInformatics LLC www.gene2drug.com 14 How do you expect your use of the following techniques used to confirm mutations in DNA to change as a result of your use of CRISPR/Cas9 technology? (Choose only one for each) 12% 21% 10% 33% 20% 30% 20% 20% 8% 10% 25% 13% 10% 10% 9% 6% 5% 13% 10% 7% 6% 5% 7% 5% 10% 7% 7% 10% 5% 6% 7% 5% 7% 9% 5% 4% 10% 4% 5% 4% 7% 10% 5% 50% 41% 40% 26% 36% 30% 49% 48% 7% 5% 5% 5% 5% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Endpoint PCR (n=135) Fluorescent microscopy (n=39) Microarray (n=20*) Next Generation Sequencing (n=84) Real-Time PCR (n=117) Digital PCR (n=20*) Sanger sequencing (n=193) Western blot (n=94) Increase by >5% Increase by 5% Increase by 4% Increase by 3% Increase by 2% Increase by 1% Stay the same Decrease
  • 15. ©2015 BioInformatics LLC www.gene2drug.com 15 PART II How to improve the CRISPR workflow and validation process (Verbatim responses)
  • 16. ©2015 BioInformatics LLC www.gene2drug.com 16©2015 BioInformatics LLC www.gene2drug.com 16 Question: A majority of scientists said ‘validation of genome editing results’ was the one area they would most like to improve. How would they propose solving this problem?
  • 17. ©2015 BioInformatics LLC www.gene2drug.com 17 Question: A majority of scientists said ‘validation of genome editing results’ was the one area they would most like to improve. How would you propose solving this problem? ©2015 BioInformatics LLC www.gene2drug.com 17 Staff Scientist, Singapore: “Ensure the targeting is unique and specific. Southern blots to confirm that only specific targeting events have occurred using probes is required to ensure there are no other insertions. Otherwise, DNA-Seq to check whole genome is a reliable albeit expensive validation method.” Senior Scientist, United Kingdom: “Good quality antibody reagents and good functional assays. Also, using other techniques such as site-directed mutagenesis and RNAi knockdown to understand the biology more fully.” Senior Scientist, United States: “The best way we have found to validate genome editing is to move to an in vivo system by SCNT and cloning. Further analysis and characterization of the genotype and phenotype of the resulting animals provide us with the most complete data sets.”
  • 18. ©2015 BioInformatics LLC www.gene2drug.com 18©2015 BioInformatics LLC www.gene2drug.com 18 Postdoctoral Fellow, United States: “Sequencing to detect the point of insertion, similar to DNA modifications, enrich for guide RNA insertions with a probe and sequence fragments that are bound to the probe.” Postdoctoral Fellow, Australia: “I don’t see any obvious possible improvement. I think the design of the target should be the main focus to avoid off target effects which are much more difficult to identify.” Laboratory Technician, United States: “A possibility is to make results "open source" so that data can be shared amongst scientists that are aiming to modify a specific gene. If they're experiencing similar results (seq data, behavior, etc.) then we may be more confident that the editing results are trustworthy. This could still be problematic as everyone might have similar off-target effects then we'd need to consider using bioinformatics to identify sequence homologies that may result in these off-target sites and seq those regions. Whole-genome seq might be another avenue to validate but that option is costly. ” Question: A majority of scientists said ‘validation of genome editing results’ was the one area they would most like to improve. How would you propose solving this problem?
  • 19. ©2015 BioInformatics LLC www.gene2drug.com 19©2015 BioInformatics LLC www.gene2drug.com 19 PhD Student, Germany: “Next gen amplicon seq would be the fastest and most accurate way to screen for modified clones. Applications which use nickase Cas9 or delivery of complex Cas9-gRNA could also minimize off targets while targeting the site of interest. According though to the application of editing, different enrichment methods could be used such as FACS for sorting out tagged proteins.” Bioengineer, France: “To ensure the validation of genome editing results, we use target specific real-time PCR or in some cases we select edited populations under selective pressure.” Laboratory Director, United States: “The only way to be sure is to do a functional protein assay or western blot, but this is impossible for all the proteins that may be altered, so using a control consisting of a bacterial or yeast gene that means nothing to the cell and showing that both cells behave the same except for the mutation.” Question: A majority of scientists said ‘validation of genome editing results’ was the one area they would most like to improve. How would you propose solving this problem?
  • 20. ©2015 BioInformatics LLC www.gene2drug.com 20©2015 BioInformatics LLC www.gene2drug.com 20 Professor, China: “Using next generation sequencing technology and PCR with specific primers.” Professor, Canada: “Next generation sequencing probably is the best method at the moment.” Principal Investigator, United States: “It would be helpful to use a genome capture and deep sequencing approach to identify edits in clones. However, this is expensive and needs to be refined to allow for a more cost effective and practical use.” Postdoctoral Fellow, Norway: “This can be simplified using specific non-toxic RNA probes.” Question: A majority of scientists said ‘validation of genome editing results’ was the one area they would most like to improve. How would you propose solving this problem?
  • 21. ©2015 BioInformatics LLC www.gene2drug.com 21©2015 BioInformatics LLC www.gene2drug.com 21 PhD Student, India: “Mutations in targeted genes were analyzed by PCR amplification and Sanger sequencing. Primers from the vicinity of the targeted regions were used for amplification of control and transformed samples. Ten cycles of amplification were performed. The amplified DNA was cloned and used for sequencing. ” Laboratory Director, United Kingdom: “You have to use a range of techniques. Science is testable, mutational research reporter assays should all give the same result. Gene transcriptome analysis would be the best: comparing gene changes with a genetic knockdown vs. CRISPR/Cas9 system should give near as identical profiling.” Staff Scientist, United States: “The simple way is end point PCR as a first diagnostic step followed by phenotypic changes.” Question: A majority of scientists said ‘validation of genome editing results’ was the one area they would most like to improve. How would you propose solving this problem?
  • 22. ©2015 BioInformatics LLC www.gene2drug.com 22©2015 BioInformatics LLC www.gene2drug.com 22 PhD Student, Philippines: “There are other ways of validating a results like we do phenotyping of lines that were produced.” Professor, Italy: “On-target and off-target genome editing should be monitored by next gen sequencing / use of selection markers could improve the identification of CRISPR/Cas9 edited cells.” Staff Scientist, United States: “Will likely remain time consuming, as single colonies need to be picked. Specific PCR, possibly followed by sequencing, still seems to be the safest method to validate a mutation genetically.” Postdoctoral Fellow, India: “I found surveyor nuclease assay to be the best assay for preliminary check. Next we can even go for NGS to check the indels.” Question: A majority of scientists said ‘validation of genome editing results’ was the one area they would most like to improve. How would you propose solving this problem?
  • 23. ©2015 BioInformatics LLC www.gene2drug.com 23©2015 BioInformatics LLC www.gene2drug.com 23 Staff Scientist, United States: “To validate, confirm the genome editing by sequencing, expression of the target gene by qRT- PCR/Western blot and finally have to perform functional assay based on the target gene.” Staff Scientist, United States: “Depends. For model organisms where off targets are less of a concern, HRMA is easy, sensitive, and works well. For human cases, deep sequencing is needed.” Professor, Finland: “Generation of KIs by inserting a defined short segments such as a series of stop codons in different frames. ” Question: A majority of scientists said ‘validation of genome editing results’ was the one area they would most like to improve. How would you propose solving this problem?
  • 24. ©2015 BioInformatics LLC www.gene2drug.com 24 Demographics (n=316) ©2015 BioInformatics LLC www.gene2drug.com 24 29% 26% 45% Geographic Region Asia-Pacific Europe North America 80% 20% Market Segment Academic Pharma/Biotech <1% 1% 1% 2% 2% 4% 8% 11% 16% 17% 17% 19% QA/QC Bioengineer Executive (CEO, VP, etc.) Laboratory Technician Other Department Head Graduate Student/Research… Lab… Post Doctoral Fellow Professor/Assistant Professor/Teacher Staff Scientist Principal Investigator Job Position 1% 1% 1% 2% 3% 3% 4% 7% 11% 14% 52% Food/Beverage Manufacturer Other Research Institute (for-profit) Contract Research Organization Hospital University Medical Center Pharmaceutical Company Government Biopharmaceutical/Biotechnology Company Research Institute/Foundation (non-profit) Academic/University Employment Sector
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  • 26. ©2015 BioInformatics LLC www.gene2drug.com 26 Interactive Market Intelligence ©2015 BioInformatics LLC www.gene2drug.com 26 Current titles: The Market for Real-Time PCR The Market for Cell-Based Assays The Market for Stem Cell Research Products The Market for CRISPR/Cas9 Genome Editing Products For more information, please visit gene2drug.com/IMI.