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You and your colleagues submitted interesting questions during the webinar, “RNA-guided genome editing with engineered Type II CRISPR/Cas9 System,” so we’ve collected and answered them here.

 
Find the answer quickly by clicking your desired question.

Q- Are the indels events that happen in a particular locus identical between the cells? Shall we not pass by a clonal step, before expending and enriching?

Q- Which is most efficient for HDR and what length is recommended for the homologous exogenous DNA: ss oligos ds oligos or large homologous arms (>1 kb) from a plasmid?

Q- To take care of the off-target issue, do we need to design more than one oligo for a gene?

Q- Several articles have discussed the non-specific activity of Cas 9. Can you comment on that?

Q- Does Cas9 cause double digestion if there are very similar DNA sequences in genome with only few differences of nucleotides?

Q- Is a 20 bp target enough for specific targeting? Will you get any off site targeting?

Q- How do you compare the CRISPR/Cas9 system with TALs in terms of specificity of the target?

Q- How can we predict mutagenesis in Danio by specific guide RNA? Downstream or upstream of the three 3' end nucleotides?

Q- What is the differences/benefits with the GeneArt® Genomic Cleavage Detection Kit and the Cel1-assay/Surveyor assay?

Q- Is your GeneArt® Genomic Cleavage Detection Kit based on the Surveyor Nuclease? Or do you use another enzyme?

Q- When you talk about point mutation, do you mean point amino acid substitution?

Q- Is it allowable to introduce big sequence s, such as GFP, or IRES-Cres et al?

Q- Your GeneArt® Genomic Cleavage Detection Kit looks a lot like the T7EI Assay, which we find to be not very specific in some cases. Can you guaranty for specific cleavage?

Q- Are there any Cas9 mutants that do not have the PAM requirement?

Q- Is the build-it-yourself, GeneArt® CRISPR Nuclease Vector kit applicable to plants yet?

Q- How does one introduce changes in the genome? Does one include mutations into the designed oligos?

Q- Must the oligos be 5' phosphorylated?

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

Q- In Slide 14, you got 99% enrichment, but only 48% cleavage, please explain?

Q- Why is there such a discrepancy in cleavage (indel %) and transfection efficiency?

Q- If a target protein is very big and multifunctional to which site is better to design sgRNA? How to check whether mutation blocks protein synthesis? How to check whether the effect is due to target mutation?

Q- How to design a guide RNA to insert a selection cassette (e.g. Neomycin)?

Q- I know that this technology can work in plant species. Is this technology available from your company in a form applicable to plants? Also, is the whole system available for purchase (i.e. do it yourself) or do you just produce vectors with the ordered target regions?

Q- Do you have any rates for editing when injecting directly into mouse embryos?

Q- You so far only use very easily accessible loci, e.g. AAVS1 or HPRT - how about "normal" loci? How does heterochromatin affect efficiency?

Q- I understand the flexibility of the 20 bp targeting crRNA, but if the PAM sequence must be present on the genomic target, doesn't this destroy this targeting flexibility?

Q- Can you explain a little bit more about the NGG PAM; what if there is not such sequence at the locus of interest?

Q- How common are the PAM loci in the human genome and does this restrict which genes can be edited?

Q- Will Life Tech offer service to generate custom cell lines using CRISPR?

Q- I noticed that the CRISPR vector you sell has the double cutting Cas9. Will you be coming out with a Cas9 vector with either the H840A nickase or D10A nickase mutations?

Q- Can we order custom made plasmids from Life Technologies with another promoter that is species specific, or cell type specific?

Q- Does U6 promoter drive expression in zebra fish embryos?

Q- Is it possible to insert a selection marker e.g. neomycin or a fluorescent marker into a pre-existing Cas9/CRISPR plasmid?

 

 

Q-Are the indels events that happen in a particular locus identical between the cells? Shall we not pass by a clonal step, before expending and enriching?

A- A clonal isolation and verification of the edited clone is advisable

 

Q- Which is most efficient for HDR and what length is recommended for the homologous exogenous DNA: ss oligos ds oligos or large homologous arms (>1 kb) from a plasmid?

A- All of them can work but for better efficiency a longer homology arm is better (at least 500bp on either side of the exogenous DNA).

 

Q- To take care of the off-target issue, do we need to design more than one oligo for a gene?

A- By careful designing of crRNA target oligos and avoiding homology with other regions in the genome one can minimize off target effect.

 

 

Q- Several articles have discussed the non-specific activity of Cas 9. Can you comment on that?

A- By careful designing of crRNA target oligos and avoiding homology with other regions in the genome one can minimize off target effect.


 

Q- Does Cas9 cause double digestion if there are very similar DNA sequences in genome with only few differences of nucleotides?

A-By careful designing of target specific guide RNA one could minimize off target effects.


 

Q- Is a 20 bp target enough for specific targeting? Will you get any off site targeting?

A-Careful designing gives you minimal off targets.

 

 

Q- How do you compare the CRISPR/Cas9 system with TALs in terms of specificity of the target?

A- Cleavage efficiency of CRISPR or TAL for one target locus can be measured using GeneArt® Genomic Cleavage Detection Kit.

 

Q- How can we predict mutagenesis in Danio by specific guide RNA? Downstream or upstream of the three 3' end nucleotides?

A- Following crRNA binding to the complementary target sequence Cas9 cleaves the target sequence ~3bp upstream of the PAM.

 

Q- What is the differences/benefits with the GeneArt® Genomic Cleavage Detection Kit and the Cel1-assay/Surveyor assay?

A- GeneArt® Genomic Cleavage Detection Kit addresses end-to-end workflow for cleavage efficiency at the locus of interest (starting from cell lysis all the way to % indel analysis).

 

Q- Is your GeneArt® Genomic Cleavage Detection Kit based on the Surveyor Nuclease? Or do you use another enzyme?

A- GeneArt® Genomic Cleavage Detection Kit addresses end-to-end workflow for cleavage efficiency at the locus of interest (starting from cell lysis all the way to % indel analysis).

 

Q- When you talk about point mutation, do you mean point amino acid substitution?

A- It can be a single base or point amino acid mutation

 

Q- Is it allowable to introduce big sequence s, such as GFP, or IRES-Cres et al?

A- Yes, it is possible.

 

Q- Your GeneArt® Genomic Cleavage Detection Kit looks a lot like the T7EI Assay, which we find to be not very specific in some cases. Can you guaranty for specific cleavage?

A- Yes, it works well with most locus.


Q- Are there any Cas9 mutants that do not have the PAM requirement?

A-No, not that we are aware of any.

 

 

Q- Is the build-it-yourself, GeneArt® CRISPR Nuclease Vector kit applicable to plants yet?

A- No, not yet.

 

Q- How does one introduce changes in the genome? Does one include mutations into the designed oligos?

A- By either oligo or transgene fragments.

 

Q-Must the oligos be 5' phosphorylated?

A- Standard oligos would suffice. You can choose Life Technologies value oligos for oligo design.

 

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

A- 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.


 

Q- In Slide 14, you got 99% enrichment, but only 48% cleavage, please explain?

A- The cells during transfection is not always synchronized population hence there is a chance that different cells are at different stage and therefore their activity may be different from cell to cell. It is possible that the cells are expressing Cas9 but the cleavage itself may not be caused due to these reasons.


 

Q-Why is there such a discrepancy in cleavage (indel %) and transfection efficiency?

A- The cells during transfection is not always synchronized population hence there is a chance that different cells are at different stage and therefore their activity may be different from cell to cell. It is possible that the cells are expressing Cas9 but the cleavage itself may not be caused due to these reasons.


 

Q- If a target protein is very big and multifunctional to which site is better to design sgRNA? How to check whether mutation blocks protein synthesis? How to check whether the effect is due to target mutation?

A- The first few exons would be best (closer to the promorter early the transcript termination). Because the gRNA efficiency depends on the accessibility of the locus as well as the chromatin structure at that location. It is advisable to design and test a few target sites. That said one could design guide RNA to as many sites as needed. Non CRISPR related mutations can be identified using non CRISPR treated sample as a control and performing a GeneArt® Genomic Cleavage Detection assay. Standard western blot analysis should tell you the level of protein.


 

Q- How to design a guide RNA to insert a selection cassette (e.g. Neomycin)?

A- Using the gRNA oligo design strategy in the manual one can design guide RNA to the locus in which you would like to insert the neomycin cassette. The cassette (neomycin) to be inserted itself can be inserted by leveraging HR in which case the neomycin cassette should contain locus specific homology arms.


 

Q- I know that this technology can work in plant species. Is this technology available from your company in a form applicable to plants? Also, is the whole system available for purchase (i.e. do it yourself) or do you just produce vectors with the ordered target regions?

A- We do not have a build-it-yourself plant specific CRISPR cataloged kit yet. But we do have custom vector design service, where researchers can send their vector design and we can build a vector with plant expression cassette and other necessary elements of their choice. Send your inquiry to CRISPR@lifetech.com and we can assist you with your project design and quote.


 

Q- Do you have any rates for editing when injecting directly into mouse embryos?

A-We haven't done these studies yet.


 

Q- You so far only use very easily accessible loci, e.g. AAVS1 or HPRT - how about "normal" loci? How does heterochromatin affect efficiency?

A- What we had shown was only part of the data; we have tested other genes as well. For example RelA, ActB etc. Accessibility and the context of the locus and its accessibility can effect the cleavage efficiency hence we advise designing and testing a few guideRNA targets.


 

Q- I understand the flexibility of the 20 bp targeting crRNA, but if the PAM sequence must be present on the genomic target, doesn't this destroy this targeting flexibility?

A- While PAM is a necessary requirement, NGG (the PAM for S. Pyogenes based CRISPR/Cas9 system) occurs quite often in a gene and hence the changes are high that you will find a NGG PAM in your gene of interest.



Q- Can you explain a little bit more about the NGG PAM; what if there is not such sequence at the locus of interest?

A- While PAM is a necessary requirement, NGG (the PAM for S. Pyogenes based CRISPR/Cas9 system) occurs quite often in a gene and hence the changes are high that you will find a NGG PAM in your gene of interest. But in its absence one could go with GeneArt® Precision TAL effector based nuclease.



Q- How common are the PAM loci in the human genome and does this restrict which genes can be edited?

A- While PAM requirement is a necessary requirement, NGG (the PAM for S. Pyogenes based CRISPR/Cas9 system) is seen quite often in a gene.



Q- Will life tech offer service to generate custom cell lines using CRISPR?

A- Yes, for all inquires contact CRISPR@lifetech.com and we can assist you with your project design and quote.



Q- I noticed that the CRISPR vector you sell has the double cutting Cas9. Will you be coming out with a Cas9 vector with either the H840A nickase or D10A nickase mutations?

A- Yes, our vectors are all double strand break inducing Cas9 with both catalytic site active. We do not have a nickase yet.



Q- Can we order custom made plasmids from Life Technologies with another promoter that is species specific, or cell type specific?

A- Yes, for all inquires contact CRISPR@lifetech.com and we can assist you with your project design and quote.



Q- Does U6 promoter drive expression in zebra fish embryos?

A- Yes.



Q- Is it possible to insert a selection marker e.g. neomycin or a fluorescent marker into a pre-existing Cas9/CRISPR plasmid?

A- Yes, if you use the current GeneArt® CRISPR nuclease vectors the respective LULLs will apply.