Feeds:
Posts
Comments

Posts Tagged ‘CRISPR/Cas and cell & molecular biology’


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 14th 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

To Follow LIVE CONFERENCE COVERAGE PLEASE FOLLOW ON TWITTER USING

Meeting #: #BostonDOT16

Meeting @: @BostonDOT

 

Overall good meeting #s:

#personalizedmedicine

#innovation

#cancer

#immunology

#immunooncology

#pharmanews

#CRSPR

#geneediting

#crisper

#biotech

 

AND FOLLOW these @

@pharma_BI

@AVIVA_1950

@BiotechNews

@CHI

@FierceBiotech

Read Full Post »


Bacterial immune system may be utilized as a tool harboring an impressive recording capacity

Curator: Larry H. Bernstein, MD, FCAP

LPBI

 

Creating a DNA Record with CRISPR

Researchers repurpose a bacterial immune system to be a molecular recording device.

By Ruth Williams | June 9, 2016     http://www.the-scientist.com/?articles.view/articleNo/46279/title/Creating-a-DNA-Record-with-CRISPR

Utilizing the bacterial CRISPR/Cas adaptive immune system, researchers at Harvard have developed a method for permanently recording molecular events in living cells, according to a report published inScience today (June 9). The system integrates specific synthetic DNA elements into the bacterial genomes in temporally-ordered arrays, which, once sequenced, can provide a readout of the bacteria’s timeline of DNA events.

“The importance of the work is in providing a proof of principle: that a fascinating bacterial immune system may be utilized as a tool harboring an impressive recording capacity,” said microbiologist Udi Qimron of Tel Aviv University who was not involved in the work.

The CRISPR/Cas system works by snipping short DNA elements from the genomes of infecting viruses, integrating those elements into the bacterium’s genome (at the CRISPR locus), and using the RNAs produced from the integrated elements to direct destruction of the corresponding virus. In essence, the bacterium keeps a DNA account of its viral foes, and uses it against them.

Integration of these viral DNA elements—or oligomers—into the CRISPR locus is nonrandom: the most recent viral elements are consistently integrated ahead of older viral elements in the array. Harvard’sGeorge Church and colleagues considered that this temporal ordering of integration could form the basis of a molecular recording device. If defined synthetic DNA oligomers could be integrated into CRISPR loci just as viral elements are, then sequencing the cells’ CRISPR loci would provide a log of which oligomers the cells had been exposed to and when, the researchers reasoned.

To test this idea, the team used an E. coli strain that contained a CRISPR DNA locus and a stripped-down version of the Cas protein machinery. The minimal machinery consisted of inducible versions of Cas1 and Cas2—enzymes required for integrating the DNA oligomers—but lacked all the Cas machinery required for virus destruction. The researchers found that, by introducing specific synthetic DNA sequences into these cells in a timed manner (different oligomers on different days, for example), the resulting sequences of the CRISPR loci did indeed accurately reflect the order in which the oligomers had been introduced.

“It’s the first demonstration of the ordered acquisition of intentionally introduced DNA sequences,” said bioengineer Adam Arkin of the University of California, Berkeley, who did not participate in the work.

Using directed evolution, the team went on to create new versions of Cas1 and Cas2 that could integrate oligomers in a subtly different and discernable way (though still temporally ordered) to that of wildtype Cas1 and 2. Putting these modified Cas enzymes under the control of a different inducer allowed the team to record DNA events in two different modes—depending on which versions of Cas1 and 2 were operational.

“Essentially, we’re measuring concentrations of nucleic acids,” said Church. “Ideally it would be messenger RNAs but in this case it is DNA. . . . This is a proof of concept on the way to other things,” he added.Church suggested, for example, that if a CRISPR/Cas system were to be combined with a reverse transcriptase—an enzyme that converts RNA to DNA—in cells or animals, it could be used to provide a record of which messenger RNAs are expressed, when.

Another possibility, suggested Arkin, is to use CRISPR/Cas-engineered bacteria to provide information about the other microorganisms present in an environment—be that the soil, the human gut, or wherever.

“[The bacteria] could kill a few neighboring [bugs], secrete an enzyme that cleaved their DNA, and express a competence system to take that DNA in,” Arkin said. “That sounds insane, but there are bacteria who do that naturally,” he added. The foreign microbial DNA could then be incorporated and logged at the bacteria’s CRISPR locus, he explained.

Such applications are as-yet distant possibilities, but the new paper, said Arkin, “conceptually sets the flag in the ground and says, ‘Here’s how we should move forward.’”

S.L. Shipman et al., “Molecular recordings by directed CRISPR spacer acquisition,” Science,doi:10.1126/science.aaf1175, 2016.

 

 

Molecular recordings by directed CRISPR spacer acquisition

Read Full Post »