Development and Characterization of a Modular CRISPR and RNA Aptamer Mediated Base Editing System

Abstract

Conventional CRISPR approaches for precision genome editing rely on the introduction of DNA double-strand breaks (DSB) and activation of homology-directed repair (HDR), which is inherently genotoxic and inefficient in somatic cells. The development of base editing (BE) systems that edit a target base without requiring generation of DSB or HDR offers an alternative. Here, we describe a novel BE system called Pin-point TM that recruits a DNA base-modifying enzyme through an RNA aptamer within the gRNA molecule. Pin-point is capable of efficiently modifying base pairs in the human genome with precision and low on-target indel formation. This system can potentially be applied for correcting pathogenic mutations, installing premature stop codons in pathological genes, and introducing other types of genetic changes for basic research and therapeutic development.

Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

“…Despite size constraints CRISPR-Cas editing devices may be delivered using AAV vectors, where nuclear targeting of viral genomes can avoid immune responses to cytosolic DNA associated with other delivery mechanisms [175][176][177][178][179]. Aptamers have been used to recruit DNA modifying enzymes for base editing [180], to improve the efficiency and reduce off-target effects of HDR-mediated gene editing [181], and to target labeled CRISPR-Cas complexes to specific subcellular locations to improve imaging techniques [182], demonstrating that small, ligand-binding RNA devices can be integrated into CRISPR-Cas systems for a variety of purposes. For therapeutic applications, particularly gene editing, CRISPR-Cas systems must be tightly regulated both temporally and spatially.…”

Combined approaches for increasing fetal hemoglobin (HbF) and de novo production of adult hemoglobin (HbA) in erythroid cells from β-thalassemia patients: treatment with HbF inducers and CRISPR-Cas9 based genome editing

“…In Table 2 a few examples are also reported discussing the newly developed prime and base editing of β-thalassemia mutations ( Liang et al, 2017 ; Zhang et al, 2022 ; Badat et al, 2023 ; Hardouin et al, 2023 ). These novel approaches are expected to limit genotoxic of the gene editing procedures, especially those due to homology-directed repair (HDR), activated following the introduction of DNA double-strand breaks (DSB) during the conventional CRISPR approach ( Liang et al, 2017 ; Komor et al, 2018 ; Zeng et al, 2020 ; Collantes et al, 2021 ; Zhang et al, 2022 ; Badat et al, 2023 ; Carusillo et al, 2023 ; Hardouin et al, 2023 ).…”

“…In addition, we have to mention that novel gene-editing strategies (for instance base-priming and base-editing), characterized by lower level of genotoxicity, are available and are expected to be extensively studied and validated in the next future ( Liang et al, 2017 ; Zipkin, 2019 ; Collantes et al, 2021 ; Zhang et al, 2022 ; Badat et al, 2023 ; Hardouin et al, 2023 ). In our opinion, this last development of gene editing protocols is of great interest to propose combined treatments based on gene editing (or multiplexed gene editing) with HbF inducers.…”

An Update of Nucleic Acids Aptamers Theranostic Integration with CRISPR/Cas Technology

“…In this system, the guide RNA (gRNA) was engineered to contain an RNA aptamer that recruits different DNA deaminases to enable the introduction of point mutations to DNA or RNA without the formation of DSBs. As a result, the Pin-point TM system demonstrated lower off-target editing than previous base editing models [ 56 ].…”