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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- 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- 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- 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- 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- How to design a guide RNA to insert a selection cassette (e.g. Neomycin)?
Q- Do you have any rates for editing when injecting directly into mouse embryos?
Q- How common are the PAM loci in the human genome and does this restrict which genes can be edited?
Q- Will you offer service to generate custom cell lines using CRISPR?
Q- Does U6 promoter drive expression in zebra fish embryos?
A- A clonal isolation and verification of the edited clone is advisable
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).
A- By careful designing of crRNA target oligos and avoiding homology with other regions in the genome one can minimize off target effect.
A- By careful designing of crRNA target oligos and avoiding homology with other regions in the genome one can minimize off target effect.
A-By careful designing of target specific guide RNA one could minimize off target effects.
A-Careful designing gives you minimal off targets.
A- Cleavage efficiency of CRISPR or TAL for one target locus can be measured using GeneArt Genomic Cleavage Detection Kit.
A- Following crRNA binding to the complementary target sequence Cas9 cleaves the target sequence ~3bp upstream of the PAM.
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).
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).
A- It can be a single base or point amino acid mutation
A- Yes, it is possible.
A- Yes, it works well with most locus.
A-No, not that we are aware of any.
A- No, not yet.
A- By either oligo or transgene fragments.
A- Standard oligos would suffice. You can choose our value oligos for oligo design.
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.
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.
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.
A- The first few exons would be best (closer to the promoter 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.
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.
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.
A-We haven't done these studies yet.
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 guide RNA targets.
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.
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 TALs Products and Services effector based nuclease.
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.
A- Yes, for all inquires contact CRISPR@lifetech.com and we can assist you with your project design and quote.
A- Yes, our vectors are all double strand break inducing Cas9 with both catalytic site active. We do not have a nickase yet.
A- Yes, for all inquires contact CRISPR@lifetech.com and we can assist you with your project design and quote.
A- Yes.
A- Yes, if you use the current GeneArt CRISPR nuclease vectors the respective LULLs will apply.