Single-stranded DNA within the Staphylococcus aureus Cas9 R-loop at the Fukutin-related protein protospacer (ssDNA in SaCas9-FKRP R-loop)

The target refers to the transiently exposed single-stranded DNA (ssDNA) segment of the Fukutin-related protein (FKRP) gene created during the formation of an R-loop by the Staphylococcus aureus Cas9 (SaCas9) protein (Ran et al., 2015, Nature). In the context of CRISPR-based genome editing, specifically base editing, SaCas9 uses a guide RNA to locate a specific protospacer sequence within the FKRP gene. Upon binding, the DNA double helix is unwound, and the non-target strand is displaced as ssDNA, making it accessible to tethered effector enzymes such as deaminases (Komor et al., 2016, Nature). This specific editing window is the site where chemical modifications, such as the conversion of cytosine to uracil or adenine to inosine, occur to correct pathogenic mutations. Mutations in the FKRP gene are primarily responsible for Limb-girdle muscular dystrophy type 2I (LGMD2I), characterized by progressive muscle wasting due to defective glycosylation of alpha-dystroglycan (Brockington et al., 2001, Am J Hum Genet). By targeting this ssDNA intermediate, base editors can precisely rewrite the genetic code without inducing double-strand breaks, offering a potential therapeutic pathway for muscular dystrophies. The interaction between the base editor and this ssDNA substrate is highly dependent on the Protospacer Adjacent Motif (PAM) and the length of the guide RNA. Therapeutic strategies focusing on this target aim to restore functional FKRP protein expression in muscle tissues.

Other names

Displaced non-target strand of FKRP protospacerSaCas9 R-loop editing windowFKRP ssDNA substrateNTS of FKRP protospacer

Targeted nucleotide deamination (A-to-G or C-to-T transition) within the displaced single-stranded DNA window of the R-loop structure.