Frequently Asked Questions:
How many constructs could constitute a single locus?
One could imagine one haplotype, two haplotypes (say one risk and one non-risk) or many, depending on the complexity of the region. We anticipate making up to 100 or more variants of a locus. In the early going, we are starting more conservatively, e.g. we are doing ~25 variants of the α-globin locus
What is the timeline?
We hope the LNB will meet sometime this fall or winter and specify 3 top tier loci, and 5 second tier loci. We would start on the three right away, and anticipate completing them over the second year of our CEGS (approximately May 2019-April 2020). If supplemental funds were obtained from NHGRI or other sources, we would add more loci (or more variants) to the list. NHGRI has expressed interest in doing more variants (500?) For at least one locus, if justified.
How far would you (the CEGS) expect to take the experiment: for example, would you put construct into ESCs/iPSCs or would we?
Would you differentiate the cells or would we? We would build and deliver the locus and its variants. For haplotype dissection we’d do a risk and protective haplotype for starters, then the variants. We could in theory do the differentiation of the ESCs/iPSCs ourselves, but would welcome collaboration on that part.
Would a region 200kb or more be practical? How would this change the likelihood for success or the timeframe for completion?
Yes we think 200 kb is practical, and it will likely be possible to do up to 500 kb. We think the assembly part up to 1 Mb is practical, the challenge is likely to be precision delivery: much larger DNAs will be more challenging in terms of both efficiency and % correct.
Can we participate in the selection or nomination of variants to be considered?
Yes! We welcome your input.
Would you consider experiments that take advantage of methods but are not focused on identifying the risk variant(s)? For example, we have cases in which we are not certain of the actual disease gene and wish to determine this.
Absolutely!
Where in the genome would you integrate the library of Assemblons for each selected locus?
Ectopic integration (e.g. at the HPRT1 locus) is currently easier than allelic replacement (where Assemblons replace one of the two alleles of the selected locus), but we are working diligently to make allelic replacement the standard method.