1:Division of Hematopoietic Innovative Therapies. Centro de Investigaciones Energéticas Medioambientales y Tecnológicas and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIEMAT/CIBERER), Madrid 28040, Spain.; 2:Advanced Therapies Unit, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid 28040, Spain.; 3:Department of Biology, ETH Zurich, Zurich, Switzerland

Base and prime editors hold several advantages in comparison to classical strategies since they do not rely on DSBs and allow the correction of specific mutations without utilizing HDR, which is inefficient in hematopoietic stem cells (HSCs). Base editing system contains a Cas9 nickase fused to a deaminase that, in combination with a sgRNA, favors the specific base conversion in a genomic target site. Cytidine base editors promote C to T base conversion and adenine base editors allow the conversion of A to G. Our results show that adenine base editing constitutes an efficient strategy to generate a specific A to G base substitution (85%) in AAVS1 locus from healthy donor HSCs. Moreover, we have demonstrated the feasibility to efficiently correct a prevalent Fanconi anemia A stop codon mutation (c.295 C>T) in patient-derived HSPCs.

Additionally, prime editing is a versatile tool that allows targeted base conversions, insertions, and deletions, consisting of a Cas9 nickase fused to a reverse transcriptase, guided to a genomic site by a prime editing guide RNA. Several optimizations have been implemented to increase initial efficiencies, such as the use of a second sgRNA that promotes the generation of a nick in the non-edited strand (PE3) or the co-expression of a dominant-negative Mismatch Repair (MMR) protein (PE5). We have demonstrated that prime editing is an efficient approach to correct the c.295C>T mutation, reaching 42% editing. Overall, our results demonstrate that base and prime editing strategies constitute realistic approaches for the treatment of FA-associated mutations.