(c) Multiplexed guide RNA expression

All of the CRIPSR/Cas9 gene drives published to date have used a single gRNA to direct site-specific homing [12,14–16]. A disadvantage of this strategy is that indels generated by NHEJ-mediated DNA repair will very likely become resistant to homing due to modification of the gRNA binding sequence and/or the associated PAM. A potential solution to this issue is to employ multiple gRNAs that cleave at several closely spaced sites across the target region [6,18]. From a practical standpoint, this could be achieved using U6 promoters from different species (to avoid recombination-mediated instability) [46] or possibly via expression of a polycistronic transcript containing multiple gRNAs using an RNA polymerase II promoter [47,48]. Instability caused by conserved gRNA sequences could potentially be avoided using diverse gRNA scaffolds [49]. Methodology for sequential gRNA expression in the germline requires development.