We have compiled a list of most-frequently asked questions from our web-based CRISPR discussion forum (discuss.genome-engineering.org) to clarify points of confusion when applying the CRISPR system (Box 3). CRISPR-Cas can be easily multiplexed to facilitate high-efficiency genome editing in mammalian cells: by using two sgRNAs, we were able to demonstrate simultaneous targeting of the human DYRK1A and GRIN2B loci at efficiencies of 65–68% for each locus in HEK 293FT cells (Fig. 5b). Likewise, a pair of sgRNAs can be used to mediate microdeletions, such as excision of EMX1 exon 3, which we genotyped by PCR at a clonal level (Fig. 5c). Note that the precise location of exon junctions can vary. We also demonstrate here the use of ssODNs and targeting vector to mediate HDR (Fig. 6a,b) with both WT and the D10A nickase mutant of Cas9 in HEK 293FT and HUES9 cells (Fig. 6c). Note that we have not been able to detect HDR in HUES9 cells by using Cas9n with a sgRNA, which may be due to low efficiency or a potential difference in repair activities in HUES9
