Your colleagues submitted interesting questions during the webinar, “Genome editing of iPSCs from Parkinson’s disease patients using TAL technology: design, detect, and quantitate,” so we’ve collected and answered them here.

 
Find the answer quickly by clicking your desired question.

Q- Why is efficiency so low? What can be done to increase efficiency?

Q- Have you begun in-vivo model study using edited iPSC cells?

Q- Will slides be available on your website later?

Q- What antibody did you use for a-syn western blot?

Q- What type of off-target changes and how did you check for them?

Q- What was the reason to choose exactly Exon 2 (in SNCA gene) for cleavage?

Q- Can this correction be used on no iPSC, e.g. neurons from PD patient?

Q- I’ve had problem is the isolation of single iPS cells. What about that in your experiments?

Q- Could using replication-defective lentivirus used to deliver the TAL plasmids? Could it be more efficient?

Q- Whether GBA mutation is associated with other neural disorders?

Q- How long does it take to synthesize the TAL?

Q- For transformation into the target organism, does TAL have to be engineered in a vector? What kind of vector is needed?

Q- Did you use selection cassettes in either the TALEN or donor templates?

Q- Was your donor template in a plasmid or was it an oligonucleotide template?

Q- Did you have a selection cassette on the donor template?

Q- On the SNCA -/-, why do you think there was still protein present at the correct size? Perhaps a mixed population, not clonal.

Q- Did you confirm absence of Sendai virus sequences in the iPSC?

Q- Have you ever encountered differences in TAL activity between the 'workhorse' cell line and your actual cell line used?

Q- Have you tested the TaqMan SNP Assay only on single clones? Would it work with all cells from a well for initial verification of TAL function or would the background be too high?

 

Q-Why is efficiency so low? What can be done to increase efficiency?

A- The efficiency is consistent with what's reported in the literature with the same technology. There are several ways one can increase efficiency, for instances, adding antibiotic selection and/or FACS sorting to enrich for transfected cells.

Q- Have you begun in-vivo model study using edited iPSC cells?

A- No

Q- Will slides be available on your website later?

A- Yes. This webinar will be available on demand

 

Q- What antibody did you use for a-syn western blot?

A- For western blotting detecting a-Syn protein, we used the Synuclein Alpha Monoclonal Antibody, Mouse (clone Syn 211) from Life Technologies, catalog number AHB0261


Q- What type of off-target changes and how did you check for them?

A- We are in the process of exploring the off-target events.

 

Q- What was the reason to choose exactly Exon 2 (in SNCA gene) for cleavage?

A-Exon 1 encodes for 5' UTR and Exon 2 contains the start of protein coding region.

 

Q- Can this correction be used on no iPSC, e.g. neurons from PD patient?

A- Unlikely. The key advantage of iPSCs is that they can be passage for a number of passages without the loss of genomic integrity and pluripotency. It's nearly impossible to obtain a number of primary neurons from PD patients. Neurons derived from PD patient iPSCs usually contain mixed cell types and are much more difficult to culture than iPSCs.

Q- I’ve had problem is the isolation of single iPS cells. What about that in your experiments?

A- As we discussed in the webinar, we employed three genomic analysis tools (In vitro cleavage assay, TaqMan® SNP genotyping and PGM sequencing) for screening and identifying colonies that are clonal population. Sometimes we had to re-pick and re-screen daughter colonies (that were plated at low density) to identify the right clones.

Q- Could using replication-defective lentivirus used to deliver the TAL plasmids? Could it be more efficient?

A- It's possible. We never tried it since we wanted to generate foot-print free isogenic lines. Lenti could leave unwanted integrations.

Q- Whether GBA mutation is associated with other neural disorders?

A- Besides PD, GBA mutations were also found in subjects with dementia with Lewy bodies.

Q- How long does it take to synthesize the TAL?

A- Typically, it takes 2 weeks to synthesize a custom GeneArt® Precision TAL

Q- For transformation into the target organism, does TAL have to be engineered in a vector? What kind of vector is needed?

A- The GeneArt® Precision TALs are delivered on Gateway entry vector. You need to perform a LR reaction to put TAL-functional domain fusion onto a destination vector. Refer to the product user manual for specifics.

Q- Did you use selection cassettes in either the TALEN or donor templates?

A- No. We did not want to generate any unwanted foot-print (exogenous sequences) in the edited line.

Q- Was your donor template in a plasmid or was it an oligonucleotide template?

A- We used purified PCR fragments (about 1 kb) that contain donor sequences. We found this is more efficient than using a plasmid. We didn't try single strand oligonucleotides.

Q- Did you have a selection cassette on the donor template?

A- No. We did not want to generate any unwanted foot-print (exogenous sequences) in the edited line.

Q- On the SNCA -/-, why do you think there was still protein present at the correct size? Perhaps a mixed population, not clonal.

A- The data shown was comparing the parental line to the heterozygous knockout line, not the homozygous knockout line.

Q- Did you confirm absence of Sendai virus sequences in the iPSC?

A- Yes, using our TaqMan® iPS Sendai Detection Kit from Life Technologies, catalog number A13640

Q- Have you ever encountered differences in TAL activity between the 'workhorse' cell line and your actual cell line used?

A- Due to higher transfection efficiency of the workhorse cell line, higher cleavage activity were detected in the workhorse cell line than in iPSCs. Since our focus was iPSCs, we did not perform detailed genomic analysis on the workhorse cell lines.

Q- Have you tested the TaqMan SNP Assay only on single clones? Would it work with all cells from a well for initial verification of TAL function or would the background be too high?

A- We have only run the TaqMan SNP assay on single clones.  It is very likely that the background will be to high for screening transfected pools.  For detecting rare events in a cell population, Taqman Mutation Detection Assays using castPCR technology is a better alternative.