Exploring the Efficiency of Multi-Target Editing with Single Base Editors in Haematopoietic Cell lines

Base editing, an extension of CRISPR-Cas9 gene editing, presents a promising strategy for correcting genetic mutations underlying inherited bone marrow failure syndromes. This approach enables ex vivo editing of haematopoietic stem cells, which can be reintroduced into patients without the risk of immune rejection. We have identified pathogenic single nucleotide mutations associated with bone marrow failure syndromes that have a nearby protospacer adjacent motif site and were amenable to adenine base editing, and designed guideRNAs to target. We performed a pooled CRISPR screen to simultaneously target these mutations in haematopoietic cell lines. This pooled strategy addresses the limitation of focusing solely on more prevalent mutations, thereby enabling the inclusion of rarer variants. Our comprehensive analysis revealed correction at 48 loci, with editing efficiencies of up to 26.22% in Jurkat cells and 19.75% in K562 cells. These findings highlight the potential of adenine base editing to correct a broad range of pathogenic variants and accelerate the development of targeted therapies for bone marrow failure syndromes.