Your colleagues had interesting questions during this successful event, so we’ve collected and answered them here. If you have any other questions, please feel free to contact our experts at firstname.lastname@example.org.
Q: What was the efficiency of LRRK2 mutation correction?
Q: What levels of transcriptional activation and repression can be achieved using this system, and is it better to use multiple TAL effectors targeted to a single gene?
Q: What is the relative efficiency of each of the 4 TAL-FokI designs against the same locus area but with different nucleotides (A, C, G, T) on the 5' end?
Q: What do the numbers at the bottom mean?
Q: To correct the LRRK2 G2019S mutation, was a linear donor fragment used, or a circular plasmid?
Q: How does this technology differ from the lentivirus-based shRNA knockdown?
Q: Do you use NN to recognize G?
Q: Do you provide optimized FokI for plant genome editing?
Q: Are the gels on slide #28 PAGE or agarose gels?
A: In the initial screen for the LRRK2 correction, 2 out of 140 colonies (1.4%) were positive for editing, using the GeneArt® Genomic Cleavage Detection Kit.
A: The example in the webinar we showed is the SOX2 gene. The activator increased SOX2 transcription 3- to 8-fold (depending on the cell type). The repressor decreased SOX2 transcription to between ½ and 1/10 of the original expression level (depending on the cell type). Typically, activation and repression levels depend on the level of normal expression and cell lines; in some cell types the gene might be highly active, and in others it might be relatively low.
A: In our tests, the 5’ base doesn’t affect the efficiency. For example, at the HPRT site, we have 4 pairs of TAL-FokI against the same area with different bases, and all 4 pairs showed similar efficiency (50–70%). Efficiency is largely governed by the spacing between the two targets. We also tested different combinations of forward and reverse TAL-FokI (for example, the forward TAL-FokI targeted to the sequences with 5’ C and the reverse TAL-FokI targeted to the sequences with 5’ G), and the efficiency is 60–70% as long as the spacing between the two targets is 15–16 bp.
A: The number below each lane on the gel picture is the indel % cleavage efficiency of TAL-FokI. It was measured by densitometry, based on the gel assay
A: It was a linear fragment generated by PCR.
A: TAL effectors directly target genomic DNA, and therefore modulate and edit at the genomic DNA level (TAL-FokI knocks out the gene). In contrast, RNAi is posttranscriptional modulation at the level of messenger RNA (achieves knockdown at the posttranscriptional level, but may not achieve knockout).
A: We offer custom GeneArt® gene codon optimization services.
A: They are 2% agarose gels (E-Gel® EX Agarose Gels).