A second research paper considerably simplified the procedure of generating complex alleles using CRISPR/Cas9 technology . Ohtsuka and colleagues reasoned that direct delivery of the CRISPR/Cas9 reagents into the mouse oviduct would be as efficient, and more effective, than microinjection and surgical transfer of zygotes . This technique would bypass all lengthy and complex procedures, from the isolation of zygotes from the oviduct, to mouse embryo transfer into recipient females. The other advantage of this method would be the reduction of the number of animals required to generate a knockout or knockin allele, since it is no longer necessary to sacrifice females for zygote collection, and the authors show that the recipient females are able to be impregnated again after their first transgenic litter.
Ohtsuka and colleagues  postulated that in situ delivery of the CRISPR/Cas9 reagents to the mouse oviduct by electroporation would allow Cas9 protein access to the zygote DNA to edit the genome. To ascertain this hypothesis, Ohtsuka and colleagues optimized the delivery protocol of CRISPR/Cas9 reagents to the oviduct and determined the optimum editing efficiency was at 0.7 dpc. The authors then hypothesized that an in vivo delivery of the CRISPR/Cas9 reagents combined with an Easi-CRISPR approach would display a similar, if not better, efficiency of editing simple or complex alleles when compared with microinjection into mouse zygotes. They tested this approach, called improved-genome editing via oviductal nucleic acid delivery (i-GONAD) (Fig. 1b), and after optimization the success of editing observed was up to 97% for straight knockout alleles and 50% for gene tags, which gives similar results to microinjected zygotes . Importantly, Ohtsuka and colleagues demonstrated the feasibility of this approach targeting various genes in multiple mouse strains. Interestingly, the recipient females could be used for multiple experiments, suggesting that it is possible to considerably reduce the number of mice needed to generate these modified alleles. Whereas the efficiency of the i-GONAD approach to generate complex alleles using CRISPR/Cas9 genome editing technologies seems remarkable and promising, the frequency of mosaicism appears to remain up to 30% of edited alleles. Future optimization of this technique, combined with replication studies from various research groups, will enable improvements to the technology, address technical hurdles, and hopefully enable the successful and efficient generation of conditional alleles using the i-GONAD technique.