CRISPR genome editing using a combined positive and negative selection system

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is a powerful genome editing tool that has revolutionized research. Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation in humans. Only a subset of these SNPs has been shown to be linked to genetic diseases, while the biological relevance of the majority remains unclear. Investigating these variants of unknown significance could provide valuable insights into their roles in biological processes, disease susceptibility, and treatment responses. While CRISPR/Cas has emerged as a transformative technology, its ability to make single nucleotide substitutions remains a significant limitation. Other techniques in single nucleotide editing, such as base editing and prime editing, offer promising possibilities to complement CRISPR/Cas systems, though they also have their own limitations. Hence, alternative approaches are necessary to overcome the limitations of CRISPR. Here, to improve the feasibility of generating single base edits in the genome, we provide a protocol that introduces a multiple expression and dual selection (MEDS) system, which, alongside CRISPR, utilizes the opposing roles of cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT) for negative selection and neomycin phosphotransferase II (NPT II) for positive selection. As a proof of concept and to demonstrate feasibility of the method, we used MEDS, along with traditional CRISPR-Cas9, to generate sickle hemoglobin by introducing a point mutation (A → T) in the sixth codon of the hemoglobin beta gene.