CRISPR/Cas therapeutic strategies for autosomal dominant disorders

Abstract

Autosomal dominant disorders present unique challenges, as therapeutics must often distinguish between healthy and diseased alleles while maintaining high efficiency, specificity, and safety. For this task, CRISPR/Cas remains particularly promising. Various CRISPR/Cas systems, like homology-directed repair, base editors, and prime editors, have been demonstrated to selectively edit mutant alleles either by incorporating these mutations into sgRNA sequences (near the protospacer-adjacent motif ["near the PAM"]) or by targeting a novel PAM generated by the mutation ("in the PAM"). However, these probability-based designs are not always assured, necessitating generalized, mutation-agnostic strategies like ablate-and-replace and single-nucleotide polymorphism editing. Here, we detail recent advancements in CRISPR therapeutics to treat a wide range of autosomal dominant disorders and discuss how they are altering the landscape for future therapies.

Figure 1. CRISPR/Cas road map for the development of autosomal dominant therapeutics.

Decision-making tree that allows researchers to determine the most appropriate therapeutic editing strategy based on responses to a series of questions. This decision tree is particularly for dividing cells and requires substantial amendments for adaptation to nondividing cells, including the removal of HDR and the inclusion of alternative approaches such as homology-independent targeted insertion (HITI) and precise integration into target chromosome (PITCH). It is also important to note that this tree is not exhaustive and parallel decisions must also be considered, such as off-targeting specificity and vector cargo limitations. Note: When deciding which CRISPR-based technology to use, it is important to evaluate each experimental design individually. Critical considerations include in vivo delivery strategies and delivery capacities, off-targeting rates, and editing efficiencies.

Figure 2. Mechanism of prime editing.

Schematic detailing critical steps of prime editing mechanism of action broken down by Cas9 activity, RT activation and function, stochastic endogenous repair mechanisms, and potential editing outcomes. (i) Protospacer hybridization between pegRNA and target DNA sequence. (ii) Engineered SpCas9 creates single-strand break in strand opposite pegRNA hybridization. (iii) Hybridization between the protospacer binding sequence (PBS) of pegRNA and newly generated 3′ flap from nickase activity. (iv) Reverse transcriptase adds nucleotides to the new 3′ end of the nicked DNA strand as directed by reverse transcription template (RTT) found adjacent to the PBS sequence. (v) An equilibrium is achieved between the unedited and edited flaps, where only one is reinserted back into DNA via endogenous DNA repair mechanisms. (vi) Insertion of 3′ flap back into the DNA and pruning of the 5′ flap by exonucleases results in the formation of a heteroduplex, where mismatch repair mechanisms determine whether the unedited strand will be remodeled in response to the edit, or whether the edit will be undone with the unedited strand as template. This process can be shifted in favor of incorporating the edit by introducing an sgRNA that nicks the unedited strand, increasing mismatch repair and improving editing efficiencies. Adapted from da Costa et al. (30).

Figure 3. SNP editing for the mutation-agnostic, allele-specific treatment of autosomal dominant disorders.

SNP editing has the potential to treat multiple mutations with a single therapeutic, relying on the selective ablation of the mutant allele by targeting CRISPR/Cas systems to allele-specific SNPs. Upstream and downstream SNPs flanking regions of high pathogenic mutations can be used to selectively create CRISPR/Cas systems and induce a targeted deletion that ablates allele expression.