Protospacer (Genomic DNA target sequence)

The protospacer is a specific segment of genomic DNA that is targeted by the CRISPR-Cas system for site-specific modification (Jinek et al., 2012, Science). It is defined by its complementarity to the spacer region of a guide RNA (gRNA) and its proximity to a Protospacer Adjacent Motif (PAM), which is required for the Cas nuclease to bind and initiate cleavage (Sternberg et al., 2014, Nature). In a therapeutic context, the protospacer serves as the precise location where genetic interventions occur, such as the disruption of a disease-causing gene or the activation of a compensatory gene. For example, in the treatment of sickle cell disease, the CRISPR-Cas9 system targets a protospacer within the BCL11A erythroid-specific enhancer to restore fetal hemoglobin production (Frangoul et al., 2021, NEJM). The interaction between the gRNA and the protospacer is the fundamental mechanism that provides the CRISPR system with its high degree of programmable specificity. However, the potential for the gRNA to bind to similar sequences elsewhere in the genome, known as off-target sites, remains a significant safety concern in clinical development (Fu et al., 2013, Nature Biotechnology). Successful targeting of the protospacer leads to double-strand breaks that are subsequently repaired by cellular mechanisms like non-homologous end joining or homology-directed repair. This target is unique because it is defined by the sequence of the therapeutic agent (the gRNA) rather than being a fixed protein receptor.

Other names

ProtospacerCRISPR target sitegRNA-binding siteTarget DNA sequence

The CRISPR-Cas complex identifies the target through PAM recognition and gRNA-DNA hybridization, leading to a site-specific double-strand break that triggers cellular DNA repair pathways (NHEJ or HDR) to achieve gene disruption or correction.