Question:

What tactics can you use to reduce off target effects when using CRISPR? See design strategy and target specificity detail on:

The Answer:

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

Question:

Can you compare the 3 methods in both cost and ease of use?

The Answer:

Please see the following technical bulletin for these details –http://www.lifetechnologies.com/content/dam/LifeTech/global/life-sciences/synthetic-biology/pdfs/GeneArt%20CRISPR%20Nuclease%20mRNA%20System.pdf

Question:

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 Answer:

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)

Question:

We are trying to label a gene of interest with a fluorescent protein do we need to serial dilute cells for analysis?

The Answer:

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.

Question:

How many target RNA sequences should we use?

The Answer:

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.

Question:

What kind of negative controls do you use when you are using CRISPR/Cas9 for knockout genes?

The Answer:

Cas9 only or gRNA only transfections will be good controls to include.

Question:

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?

The Answer:

Our current products are mostly for mammalian systems. Attached are couple references for CRISPR mediated genome editing in plants:

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

Question:

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

The Answer:

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).

Question:

I am trying to cleave 2 targets for multiplex genome engineering, which method would you recommend I use.

The Answer:

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).

Question:

What is the best way to verify that clones have the correct insert prior to full sequence verification?

The Answer:

Is this question regarding clonally isolated edited cell? If so one could send the locus specific PCR generated from this specific clone for sequencing.

Question:

Can I get assistance with CRISPR gRNA design in order to eliminate off-target effects?

The Answer:

Yes, complete the CRISPR String DNA order form and submit to GeneArtSupport@lifetech.com

Question:

Can the new Cas9 mRNA be used for microinjections?

The Answer:

Yes, use the complete RNA format with the T7 GeneArt® CRISPR String DNA in vitro transcribed with our MEGAshortscript™ T7 Transcription Kit

Question:

How big is TAL? Or what’s the size of TALs?

The Answer:

3.3 Kb TALDNA

Question:

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

The Answer:

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 economically and clinically valuable products

Question:

What are the advantages of the GeneArt® CRISPR-Cas9 mRNA format compared to your existing GeneArt® CRISPR Nuclease all-in-one vector format?

The Answer:

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.

Question:

How specific is TAL? Off-site targeting?

The Answer:

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.

Question:

Can Cas9 mRNA be used to edit plant genomes?

The Answer:

There is no available protocol for Cas9 transfection into plants however we suggest GeneArt® Precision TALs for editing plant genomes.

Question:

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?

The Answer:

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.

Question:

How efficient is the GeneArt® CRISPR-Cas9 mRNA format compared to the GeneArt® CRISPR Nucleases all-in-one OFP or CD4 format?

The Answer:

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.

Question:

What’s the advantage of knock out by TAL or CRISPR compared to vector-based stable shRNA?

The Answer:

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.