Theoretically, SpCas9 permits finding a suitable target site every 8 nucleotides on an average throughout the genome. However, some genomic regions are not easily targetable by SpCas9 due to a high A/T content. Several naturally occurring orthologs of Cas9 with alternative PAM specificities have been identified and adopted for gene editing; however, many of these have even more limiting PAM requirements. • Packaging and delivery: In vivo delivery of CRISPR/Cas9 into mammalian cells is generally accomplished using viral vectors. AAVs remain the preferred choice due to their low immunogenicity and high transduction efficiency. However, AAVs have limited packaging capacity and hence, it is difficult to package the genes encoding most used Cas9 (SpCas9) and its associated sgRNA into a single AAV vector unless compact promoters are used. Another limiting factor for most gene editing components is their safe, efficient, and targeted delivery to the specific organ or tissue.