Homologous recombination strategy for introduction of the polyQ expansion in HTT exon 1 into wild type cells.

(a) Homologous donor containing a neomycin resistance cassette 1.5 kb upstream of exon 1 as described previously was used as a donor. Cas9 guide RNA (gRNA) sites were designed near the translational start site of the gene. HTT gRNA sequences were designed based upon criteria described previously. (b) Experimental design for treatment of 293F cells with Cas9 constructs. Cells were lipofected at day 0, placed on selection at day 1 and remained on selection until the cells were harvested at day 18. (c) At day 18 cells were fixed and stained with methylene blue/methanol and scanned on a flatbed scanner. Resulting scans were analyzed with the colony counting function in ImageJ. (d) There was a significant increase in colonies in all cells treated with Cas9, HTT gRNA and donor. This increase was most pronounced in cells expressing WT Cas9 and HTT specific gRNAs. The increase in colony number was donor-dependent.