Choosing the Right Genome Editing Technology for your Application and Research

Sponsored by: Life Technologies
Session ends: August 15th, 2014, 3:00pm MST
Answers by: Namritha Ravinder, Ph.D, Synthetic Biology Division, Thermo Fisher Scientific

Introduction


Genome editing —precise, site-specific DNA modification —can now be achieved through the use of chimeric protein constructs that consist of a sequence-specific binding protein linked to a non-specific endonuclease that cleaves DNA a predictable distance from the binding site. The DNA-binding domains of transcription activator–like (TAL) effectors are known and programmable, and that knowledge can be used to create customized proteins that bind specifically to virtually any desired DNA sequence. Recently, clustered regulatory interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated (Cas) endonucleases, have also been used for genomic editing. Like the chimeric TAL effector nucleases (TALENs), these RNA-guided endonuclease (RGEN) systems also have modular DNA recognition and cleavage functions—by engineering the DNA-recognition components, the endonuclease components of CRISPR/Cas systems can be targeted with high specificity to cut any genomic sequence desired.

How does genome editing fit into your research, now and in the future?

Deciding which technology to use for your research may not be as easy as you think. There are many factors you should consider — what application will you be using, what cell type, desired modification, target sequence constraint’s etc.

This Ask the Expert session is sponsored by Life Technologies and will be hosted by Namritha Ravinder, Ph.D, Staff Scientist, Synthetic Biology Division, Thermo Fisher Scientific. Namritha is the R&D lead for genome editing product development within the Synthetic Biology group at Thermo Fisher Scientific. Her team focuses on building tools and products for Genome and Cell Engineering applications. Prior to this role, she was part of the Synthetic Biology Custom services team and was involved in designing customized workflows that included a variety of Life Technologies platforms including cDNA library generation, Cloning, Next Generation Sequencing, RNAi and TaqMan qPCR. She has a Masters degree in Biochemistry from India and a Doctoral degree in Plant Molecular Biology and Biotechnology from University of Alabama. She did her Postdoctoral research at Children’s Hospital in Los Angeles where she was involved in studying the Mechanism of “Enhancement of virus release” by HIV accessory proteins Vpu and Env as well as identifying their respective host cellular restriction factors.

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This session is sponsored by
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Questions & Answers

We recently began working with yeast cells, (our first time working in plants) and we are looking for an effective way to insert a gene of interest and also include a promoter gene. Do you have any advice on where we should start?

Our current products are mostly for mammalian systems. Attached are couple references for CRISPr mediated genome editing in plnats: http://www.ncbi.nlm.nih.gov/pubmed/24112467; http://www.ncbi.nlm.nih.gov/pubmed/24854982; http://www.ncbi.nlm.nih.gov/pubmed/24836556; http://www.ncbi.nlm.nih.gov/pubmed/23958582» Read More

I am concerned about the possible off target effects of long term Cas9 expression. Can you direct me to any research being done on this? Thanks

You can express Cas9 transiently and avoid integration into the genome. Published work with Cas9 nickase and dimeric RNA guided Fok1 nucleases have shown relatively more specificity (http://www.nature.com/nbt/journal/v32/n6/full/nbt.2908.html; http://zlab.mit.edu/assets/reprints/Ran_FA_Cell_2013.pdf). More recently it has been shown that direct transfection of Cas9 protein works “almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects” […]» Read More

Are TAL-meditated KO or KI strains considered to be GMO?

KO and KI involves editing the native genetic code by either mutating or deleting a encoded message or inserting a new piece of information at a desired site. Although this does manipulate the native genetic information, this technology when used in a responsible manner has very useful applications like engineering yeasts for insulin production or […]» Read More

What is the success rate or percentage efficiency for simultaneous delivery of all different target specific gRNA’s into a given cell? Or what percentage of cells in a given population gets all different gRNA?

We have not done clonal isolation to assess the cleavage efficiency to test the % population that has all gRNAs in a given cell. We have done multiplexing with up to 4 targets transfected simultaneously and, based on GeneArt® Genomic Cleavage Detection assay, we see 50% of the population modified with any one gRNA compared […]» Read More