Leaders in Pharmaceutical Business Intelligence (LPBI) Group

LIVE 9/19 10:40 – noon CRISPR Engineering Lymphoma Lines & Will Interference from CRISPR Silence RNAi? CHI’s 2nd Annual Symposium CRISPR: Mechanisms and Applications @ CHI’s 14th   Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

 

CHI’s 2^nd Annual Symposium CRISPR: Mechanisms and Applications @ CHI’s 14th 

Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

http://www.discoveryontarget.com/

http://www.discoveryontarget.com/crispr-therapies/

Meeting #: #BostonDOT16

Meeting @: @BostonDOT

 

Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a

Media Partner of CHI for CHI’s 14^th Annual Discovery on Target taking place September 19 – 22, 2016 in Boston.

In Attendance, streaming LIVE using Social Media

Aviva Lev-Ari, PhD, RN

Editor-in-Chief

http://pharmaceuticalintelligence.com

 

10:40 Vignettes From the Bench: CRISPR Engineering Lymphoma Lines

Arthur L. Shaffer, III, Ph.D., Staff Scientist, Laboratory of Dr. Louis Staudt, Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health

CRISPR/CAS9 technology is a powerful tool that permits the easy exploration of human genetics using cell line models. Our lab focuses on understanding the wiring of lymphoma cells in an effort to discover new therapeutic options. I will relate some lessons we’ve learned as we adapt CRISPR/Cas9 to the study of lymphoma.

COMMENTS by Stephen J Williams, PhD

 

 

10:40 Vignettes From the Bench: CRISPR Engineering Lymphoma Lines

Arthur L. Shaffer, III, Ph.D., Staff Scientist, Laboratory of Dr. Louis Staudt, Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health

 

RNAi screens first used to identify some of the genetic features of lymphomas

 

Generating the system

• Produce Cas9 viral particles
• Transduce cells 3 to 4 times with cas9
• Validate select pools
• Takes long time

 

Increase efficiently of transductions

• GFP tag cells and with selection marker to monitor and select; increase the selectable marker and get a more enriched pool
• Some cell lines behave well but some cell lines hate lentivirus so maybe electroporation
• Test toxiicity with sgRNA targeting RPL6
• Target a surface marker with sgRNA to validate the function of system (then can do by FACS) eg like CD20 or ICAM (CD54)
• They used a DOX inducible Cas9 but some cells can be leaky (hidden activated promoter?)
• Important to characterize all lines but he says keep even the leaky lines they might be useful down the line

 

Is AASV a good locus to use (as a control; low toxicity)?

 

• On chromosome 19 but has lots of copies of other genes and you could get close to off target “Genomic copy number dictates a gene-independent cell line response to CRSPR”
• Know your system!

 

• The level of big data with CRSPR screens is in the order of magnitute of 100s compared to other transcription analyses like expression arrays
• Need huge servers
• Need a good programmer to use the software packages as they are not plug and play SO A GOOD BIOCURATION SYSTEM IS NEEDED

 

Question/Comment:  Many cancer cell lines have different DNA repair systems active and may be why some are good lines to use and some bad but this has not been characterized yet.

 

Question/Comment:  Broad Institute has reported the CRSPR HIV system and remove 60 plus copies of a retrovirus.

 

Question: can you use p glyoprotien for a positive control?

 

Use these Twitter #

#geneediting

#CRISPR

#Cas9

#NIH

#CMT

#drugdiscovery

#pharmanews

#CRSPR

#genomics

 

 

11:10 PANEL DISCUSSION: Will Interference from CRISPR Silence RNAi?

 

Moderator: Scott Martin, Ph.D., Group Lead, Functional Genomics, Genentech Inc.

 

MMSK: New CRSPR systems being developed are looking positve for the field.  The predisigned systems are well defined but you can’t modify them in screens.  CRSPRi looks interesting but not wide penetrance of people using it in the field.  They would like to be able to offer packets to clients but many genes will be better served by using siRNA.  Not going to retire siRNA any time soon nor shRNA (some want knockdown and also knockout).

 

NIH:  CRSPR screens are showing better results than shRNA.  Off target effects are better minimized.  Drawback of CRSPRi is the limited coverage of the genome.  Must have a strong phenotype to get high coverage. Useful to get hits out of shRNA screening and then use the CRSPR as a method to interogate a pathway.  RNAi works better though to look at pathway members.

 

Abbvie:  cas9 is not easy to deliver.  So sometimes siRNA might just be good enough.  However for screens most important is to minimize off target

 

NIH:  CRSPR best way to generate cell lines. Combinitorial chemistry was thought to be panacea but now it is used to optimize a lead.

 

It is a tricky system that can turn systems on that were intitally off.  And there were alot of cell optimization problems so for NIH from NCI 60 panel maybe 25 behave and the rest are problematic.
NIH has used germinal cell lines as a surrogate for primary lines and used shRNA.  

 

Diffuse Large B Cell Lymphoma @ NIH, NCI

• 500,000 people affected in the US, increases with age, refractory 50%, diagnostic biopsies are undistinguishable

Gene expression profile

• Activated B Cells
• Germinal line – aggressive

Genomic Assult of Cancer

Making lines that effectively express Cas9 – in ~3 months

1. produce concentrate virus for Cas9
2. Transduce selective Blasticinin – Select cells efficiently – place in 8pt Blast
3. Making lines that cuts well

• 28 pass for cells for single cell cloning
• 17 effective clones – Transduce cells with sgRNA (gfp+) targeting a surface marker line CD20 and CD54 Cutters vs Partial cutters vs Leaky
• ABC  Amplification – TMD8 – false positive toxicity
• Big DATA — Gene expression, RNA sequencing, Terabyte Servers needed for CRISPR – Cas9

11:10 PANEL DISCUSSION: Will Interference from CRISPR Silence RNAi?

Moderator: Scott Martin, Ph.D., Group Lead, Functional Genomics, Genentech Inc.

Participants: Session Speakers

Each speaker will spend a few minutes sharing their viewpoints and experiences using CRISPR/Cas system for functional screening and complementing it with RNAi screening. Attendees will have an opportunity to ask questions and share their opinions.

Two sources of Noise in identifying the Cutting Point of CRISPR – Cas9 are coming from:

• Guideline design
• Cell amplification