The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is the latest RNA-guided, endonuclease tool in genome editing which allows for very specific genomic disruption and replacement. ;;The Cas9 nuclease serves to unwind the genomic DNA duplex next to conserved protospacer adjacent motifs (PAMs) and homes in on its target sequence, which is recognized by a complementary single-guide RNA. The resulting double-stranded break gets repaired by the non-homologous end joining (NHEJ) pathway, leading to a disruption in the open reading frame of the targeted gene. Alternatively, by supplying a suitable repair template, virtually any desired point mutation can be introduced at the break point via homology-directed repair (HDR).;;The Cas9 nuclease from the bacteria Streptococcus pyogenes, abbreviated spCas9, is the most commonly used Cas9 variant. The reason for spCas9 popularity is two-fold. First the spCas9 PAM sequence is 5’-NGG, which is highly abundant in the genome allowing virtually any gene to be targeted. The spCas9 enzyme also has on average a higher efficiency in vivo compared to other variants.
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