Given that such extensive validation would likely increase the time and cost involved in functional studies, and potentially preclude high-throughput analyses, a few alternative strategies might be employed. First, multiplexing many gRNAs in a single vector might increase the likelihood, not only that at least one gRNA is functional, but also that all gRNAs are expressed in each cell, thereby achieving more consistent effects than might be obtained using a pool of gRNA vectors. Second, because IVT gRNAs can substitute for lentiviral delivery, this would reduce the time and costs associated with vector design, construction, and viral packaging, while still being compatible with a multiplex strategy whereby many IVT gRNAs targeting the same gene could be transfected simultaneously. Our hope is that a threshold number of gRNAs will be empirically established, such that when they are combined in a multiplex strategy, there is a high degree of confidence that effective transcriptional modulation across all individuals and cell types of interest will be achieved.
