Leaders in Pharmaceutical Business Intelligence (LPBI) Group

LIVE 9/21 8AM to 10:55 AM Expoloring the Versatility of CRISPR/Cas9 at 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/

Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a

Media Partner of CHI for CHI’s 14^th Annual Discovery on Targettaking 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

#BostonDOT16

@BostonDOT

 

COMMENTS BY Stephen J Williams, PhD

EXPLORING THE VERSATILITY OF CRISPR/Cas9

 

8:00 Chairperson’s Opening Remarks

TJ Cradick , Ph.D., Head of Genome Editing, CRISPR Therapeutics

 

@CRISPRTX

 

8:10 Functional Genomics Using CRISPR-Cas9: Technology and Applications

Neville Sanjana, Ph.D., Core Faculty Member, New York Genome Center and Assistant Professor, Department of Biology & Center for Genomics and Systems Biology, New York University

 

CRISPR Cas9 is easier to target to multiple genomic loci; RNA specifies DNA targeting; with zinc finger nucleases or TALEEN in the protein specifies DNA targeting

 

• This feature of crisper allows you to make a quick big and cheap array of a GENOME SCALE Crisper Knock out (GeCKO) screening library
• How do you scale up the sgRNA for whole genome?; for all genes in RefSeq, identify consitutive exons using RNA-sequencing data from 16 primary human tissue (alot of genes end with ‘gg’) changing the bases on 3’ side negates crisper system but changing on 5’ then crisper works fine
• Rank sequences to be specific for target
• Cloned array into lentiviral and put in selectable markers
• GeCKO displays high consistency betweens reagents for the same gene versus siRNA; GeCKO has high screening sensitivity
• 98% of genome is noncoding so what about making a library for intronic regions (miRNA, promoter regions?)
• So you design the sgRNA library by taking 100kb of gene-adjacent regions
• They looked at CUL3; (data will soon be published in Science)
• Do a transcription CHIP to verify the lack of binding of transcription factor of interest
• Can also target histone marks on promoter and enhancer elements
• NYU wants to explore this noncoding screens
• sanjanalab.org

 

@nyuniversity

 

8:40 Therapeutic Gene Editing With CRISPR/Cas9

TJ Cradick , Ph.D., Head of Genome Editing, CRISPR Therapeutics

 

NEHJ is down and dirty repair of single nonhomologous end but when have two breaks the NEHJ repair can introduce the inversions or deletions

 

• High-throughput screens are fine but can limit your view of genomic context; genome searches pick unique sites so use bioinformatic programs  to design specific guide Rna
• Bioinformatic directed, genome wide, functional screens
• Compared COSMID and CCTOP; 320 COSMID off-target sites, 333 CCtop off target
• Young lab GUIDESeq program genome wide assay useful to design guides
• If shorten guide may improve specificity; also sometime better sensitivity if lengthen guide

 

• Manufacturing of autologous gene corrected product ex vivo gene correction (Vertex, Bayer, are partners in this)

 

 

They need to use a clones from multiple microarrays before using the GUidESeq but GUIDEseq is better for REMOVING the off targets than actually producing the sgRNA library you want (seems the methods for library development are not fully advanced to do this)

 

The score sometimes for the sgRNA design programs do not always give the best result because some sgRNAs are genome context dependent

9:10 Towards Combinatorial Drug Discovery: Mining Heterogeneous Phenotypes from Large Scale RNAi/Drug Perturbations

Arvind Rao, Ph.D., Assistant Professor, Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center

 

Bioinformatics in CRISPR screens:  they looked at image analysis of light microscopy of breast cancer cells and looked for phenotypic changes

 

• Then they modeled in a small pilot and then used the algorithm for 20,000 images (made morphometric measurements)
• Can formulate training statistical algorithms to make a decision tree how you classify data points
• Although their algorithms worked well there was also human input from scientists

Aggregate ranking of hits programs available on web like LINKS

 

@MDAndersonNews

 

10:25 CRISPR in Stem Cell Models of Eye Disease

Alexander Bassuk, M.D., Ph.D., Associate Professor of Pediatrics, Department of Molecular and Cellular Biology, University of Iowa

 

Blind athlete Michael Stone, biathlete, had eye disease since teenager helped fund and start the clinical trial for Starbardt disease; had one bad copy of ABCA4, heterozygous (inheritable in Ahkenazi Jewish) – a recessive inheritable mutation with juvenile macular degeneration

• Also had another male in family with disease but he had another mutation in the RPGR gene
• December 2015 paper Precision Medicine: Genetic Repair of retinitis pigmentosa in patient derived stem cells
• They were able to correct the iPSCs in the RPGR gene derived from patient however low efficiency of repair, scarless repair, leaves changes in DNA, need clinical grade iPSCs, and need a humanized model of RPGR

@uiowa

10:55 CRISPR in Mouse Models of Eye Disease

Vinit Mahajan, M.D., Ph.D., Assistant Professor of Ophthalmology and Visual Sciences, University of Iowa College of Medicine

• degeneration of the retina will see brown spots, the macula will often be preserved but retinal cells damaged but with RPGR have problems with peripheral vision, retinitis pigmentosa get tunnel vision with no peripheral vision (a mouse model of PDE6 Knockout recapitulates this phenotype)
• the PDE6 is linked to the rhodopsin GTP pathway
• rd1 -/- mouse has something that looks like retinal pigmentosa; has mutant PDE6; is actually a nonsense mutation in rd1 so they tried a crisper to fix in mice
• with crisper fix of rd1 nonsense mutation the optic nerve looked comparible to normal and the retina structure restored
• photoreceptors layers- some recovery but not complete
• sequence results show the DNA is a mosaic so not correcting 100% but only 35% but stil leads to a phenotypic recovery; NHEJ was about 12% to 25% with large deletions
• histology is restored in crspr repaired mice
• CRSPR off target effects: WGS and analyze for variants SNV/indels, also looked at on target and off target regions; there were no off target SNVs indels while variants that did not pass quality control screening not a single SNV
• Rhodopsin mutation accounts for a large % of patients (RhoD190N)
• injection of gene therapy vectors: AAV vector carrying CRSPR and cas9 repair templates

CAPN mouse models

• family in Iowa have dominant mutation in CAPN5; retinal degenerates
• used CRSPR to generate mouse model with mutation in CAPN5 similar to family mutation
• compared to other transgenic methods CRSPR is faster to produce a mouse model

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