Basics of Design: Knockout and Knock-in Design Using TAL Effectors and CRISPRs
Complete Q&A from the webinar
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Q: Can establishing a point mutation using CRISPR work if the closest gRNA pair is about 30 bp (1st gRNA) and 70 bp (2nd gRNA) away from the point mutation site?
A: 30 bases or even 70 bases can work OK. We always recommend using strategies to make sure your donor molecule does not get targeted by the editing tool. Another thing to keep in mind is to use at least 500 bases (or more) of homologous sequences flanking the mutation site.
Q: Do the TAL effectors and CRISPRs have preferences for specific sequences or specific gene types, for example transcription factors or structural genes?
A: Both TAL effectors and CRISPRs are guided through recognition of their target sites’ DNA sequences. Based on this, they should not exhibit any preference for any type of genetic element. They should work fine on genes of all types, as well as intergenic, intronic, promoter, and other DNA elements.
Q: What is the minimum effective cell input needed to detect cleavage using the GeneArt® assay? Does it work under non-optimized transfection conditions?
a. The cell input requirement varies and depends on the cell line and locus of interest. In some good cell lines like HEK293, you can get a good PCR product with as few as 5,000 cells (refer to the GeneArt® Genomic Cleavage Detection Kit user manual for more information).
b. To answer your second question, the Genomic Cleavage Detection assay can work under non-optimized conditions in cases where the CRISPR/TAL under question happens to cut with very high efficiency. Since the CRISPR/TAL efficiency is experimentally determined, we always recommend that you work under optimal transfection conditions.
Q: What transfection method is used, and what is a typical or good efficiency?
A: Every cell line is unique and requires optimized transfection conditions. We suggest that you look at the efficiencies using both methods: lipid-based and electroporation. To help you choose the optimal reagent for your experiment, refer to our transfection reagent selection guide for details and guidelines based on cell type.
Q: What is the difference between the gene disruption and mutation? Is the gene disruption a type of gene mutation?
A: Gene disruption involves introducing a frameshift mutation via the NHEJ repair pathway of eukaryotic cells. “Mutation” typically refers to creating a point mutation in the DNA, for example to achieve an amino acid change within the coding sequence of the gene.
Q: Was your donor DNA circular or linear?
A: Our DNA was circular. We have had good success with both of these formats.
Q: Do you have concerns about RNA stability?
A: RNA stability is important. Our CRISPR nuclease mRNA contains several proprietary modifications, including an ARCA cap and others that enhance its stability inside the cells. That said, we are not concerned about keeping mRNA stable for a long period of time. The goal is to create the cleavage at the desired target site. Once that job is done, there is no longer any need for more mRNA or resulting protein.
Q: How can one improve the cleavage efficiency of these tools?
A: Several different methods can be used to enhance the efficiency of a tool—for example, changing from plasmid to mRNA tends to help with the cleavage efficiency.
Q: Have you tried engineering amino acid substitutions in polyploid cell lines using CRISPR, and if so, what was your success rate?
A: Thus far we have not attempted to work with polyploid cells. We usually work with diploid cells and frequently achieve modification in both alleles.
Q: Which transfection reagent do you recommend for transfection of human ES cells with Cas9 mRNA and sgRNA?
A: We have had good results with Lipofectamine® MessengerMAX™ reagent for iPSCs
Q: What reagent do you use to transfect the stem cells?
A: We have had good results with Lipofectamine® MessengerMAX™ reagent for iPSCs.
Q: Can we make frameshift mutations using TALs and CRISPR/Cas?
A: Yes, the goal of the initial gene disruption workflow I showed was to introduce a frameshift mutation.