Production of Cell Models with Differential Zygosity Using CRISPR Base Editors

CRISPR base editors have revolutionized the ease with which single-nucleotide gene editing can be performed in cell and organismal models. Unlike conventional CRISPR/Cas9 gene editing, which induces a double-strand DNA break and relies on endogenous homology-directed repair (HDR) or non-homologous end joining (NHEJ) repair, base editors only induce a single-strand nick and exploit the mismatch repair (MMR) system for repair. In many instances, this results in fewer off-target effects and less cytotoxicity. Moreover, because base editors use an alternative repair mechanism, the zygosity of repair is often distinct from that of traditional HDR-based editing. Here, we provide a detailed protocol for performing gene editing using an adenine base editor (ABE) to induce a single A-G transition mutation in a gene of therapeutic relevance. We also illustrate how to sort and isolate cells with differential zygosity state, enabling the production of cell models suited to the study of both dominant and recessive mutations. This workflow can easily be adapted to the use of other variants of base editors (e.g., cytosine base editors) and can be employed in diverse cell lines.