Construction and Stability of All-in-One Adenovirus Vectors Simultaneously Expressing Four and Eight Multiplex Guide RNAs and Cas9 Nickase.

CRISPR/Cas9 technology is expected to offer novel genome editing-related therapies for various diseases. We previously showed that an adenovirus vector (AdV) possessing eight expression units of multiplex guide RNAs (gRNAs) was obtained with no deletion of these units. Here, we attempted to construct "all-in-one" AdVs possessing expression units of four and eight gRNAs with Cas9 nickase, although we expected obstacles to obtain complete all-in-one AdVs. The first expected obstacle was that extremely high copies of viral genomes during replication may cause severe off-target cleavages of host cells and induce homologous recombination. However, surprisingly, four units in the all-in-one AdV genome were maintained completely intact. Second, for the all-in-one AdV containing eight gRNA units, we enlarged the E3 deletion in the vector backbone and shortened the U6 promoter of the gRNA expression units to shorten the AdV genome within the adenovirus packaging limits. The final size of the all-in-one AdV genome containing eight gRNA units still slightly exceeded the reported upper limit. Nevertheless, approximately one-third of the eight units remained intact, even upon preparation for in vivo experiments. Third, the genome editing efficiency unexpectedly decreased upon enlarging the E3 deletion. Our results suggested that complete all-in-one AdVs containing four gRNA units could be obtained if the problem of the low genome editing efficiency is solved, and those containing even eight gRNA units could be obtained if the obstacle of the vector size is also removed.