GO: a functional reporter system to identify and enrich base editing activity.

ABSTRACT: Base editing (BE) is a powerful tool for engineering single nucleotide variants (SNVs) and has been used to create targeted mutations in cell lines, organoids and animal models. Recent development of new BE enzymes has provided an extensive toolkit for genome modification; however, identifying and isolating edited cells for analysis has proven challenging. Here we report a 'Gene On' (GO) reporter system that indicates precise cytosine or adenine base editing in situ with high sensitivity and specificity. We test GO using an activatable GFP and use it to measure the kinetics, efficiency and PAM specificity of a range of new BE variants. Further, GO is flexible and can be easily adapted to induce expression of numerous genetically encoded markers, antibiotic resistance genes or enzymes, such as Cre recombinase. With these tools, GO can be exploited to functionally link BE events at endogenous genomic loci to cellular enzymatic activities in human and mouse cell lines and organoids. Thus, GO provides a powerful approach to increase the practicality and feasibility of implementing CRISPR BE in biomedical research.

SUBMITTER: Katti A 

PROVIDER: S-EPMC7102966 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature