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.
TAL and CRISPR edits the genome and hence more efficient. In case of stable shrNA the expressed shRNA works at the level of transcripts hence the effect of knock down in gene expression depends on the level of expression of shRNa the activity of the promoter at the locus where the shRNa is stably integrated as well as the ratio of shRNa to mRNa transcripts.
How efficient is the GeneArt® CRISPR-Cas9 mRNA format compared to the GeneArt® CRISPR Nucleases all-in-one OFP or CD4 format?
In most cell types we have tested complete RNA format demonstrates equal or higher cleavage efficiency compared to plasmid format. We recommend optimizing Cas9 to gRNA dosage for maximizing efficiency for your specific cell type.
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 to singleplex.
There is no available protocol for Cas9 transfection into plants however we suggest GeneArt® Precision TALs for editing plant genomes.
Recent paper by Prashant Mali in Nature Biotechnology shows that TALs are tolerant to 1-2 mismatches and less to large majority of 3 bp mismatches.
What are the advantages of the GeneArt® CRISPR-Cas9 mRNA format compared to your existing GeneArt® CRISPR Nuclease all-in-one vector format?
Cas9 RNA format circumvents the need for cloning, has a smaller payload size, allows Cas9 to gRNA dosage optimization, easier for multiplexing, microinjection, and has no promoter constraints.
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 engineering cells for more economiocally and clinically valuable products.
3.3 Kb TALDNA
Yes, use the complete RNA format with the T7 GeneArt® CRISPR String DNA in vitro transcribed with our MEGAshortscript™ T7 Transcription Kit
Yes, complete the CRISPR String DNA order form and submit to GeneArtSupport@lifetech.com
What is the best way to verify that clones have the correct insert prior to full sequence verification?
Is this question regarding clonally isolated edited cell? If so one could send the locus specific PCR generated from this specific clone for sequencing.
I am trying to cleave 2 targets for multiplex genome engineering, which method would you recommend I use.
Use 2 gRNA simultaneously co-transfected with Cas9. If using a robust work horse cell line like 293 you could directly use the U6 synthetic DNA fragment. For more difficult to transfect cell lines we recommend using complete RNA format (Cas9 mRNA+ in vitro transcribed gRNA synthesized from T7 promoter containing DNA template).
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” (http://genome.cshlp.org/content/early/2014/04/02/gr.171322.113).
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
Cas9 only or gRNA only transfections will be good controls to include.
We recommend testing more than 1 up to three targets to ensure you have a gRNA tat gives best cleavage efficiency for the locus of interest.
We are trying to label a gene of interest with a fluorescent protein do we need to serial dilute cells for analysis?
If the fluorescent tagged gene is constitutively expressed then following knock-in experiments you can enrich cells that express the fluorescent marker. Following enrichment to get a pure clonal population you can serially dilute to get single cell derived clones.
I have heard that certain editing methods work better with certain cell lines, if so how do you know which method to use with your cell line?
The key is to know the best format of nucleic acid and the delivery reagent that is ideal for your cell type. For most cell lines we have tested complete CRISPR RNa format (Cas9 mRNA + in vitro transcribed gRNa) with Lipofectamine messenger max works well. Please note certain cell types electroporation works better. Refer to tranfection reagent guidelines (http://www.lifetechnologies.com/us/en/home/life-science/cell-culture/transfection/transfection-support.html)
Please see the following technical bulletin for these details -
What tactics can you use to reduce off target effects when using CRISPR? See design strategy and target specificity detail on:
To answer this question, please see design strategy and target specificity detail on: http://www.lifetechnologies.com/content/dam/LifeTech/global/life-sciences/synthetic-biology/pdfs/0913/CRISPR%20Technical%20Product%20Bulletin%20(Global)_FLR.pdf