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.

BEAR reveals that increased fidelity variants can successfully reduce the mismatch-tolerance of adenine but not cytosine base editors

“…One solution for increasing the e ciency of base editing without generating nick-induced indels would be the enrichment of those cells that contain dABE-and dCBE-edited bases, using a marker. Unfortunately, the markers that have been developed so far to enrich CBE-or ABE-edited cells exclusively employ nickase SpCas9 12,13,14,15,16,17 . One of the objectives of our study has been to develop a marker that is sensitive enough to enable high e ciency base editing by enriching dABE-and dCBE-edited cells, without intentionally nicking the DNA.…”

“…Recently, a few approaches have been reported that allow the employment of uorescence-based assays. These assays are based on the installation or alteration of a start or stop codon 16,17 , or they rescue a disruptive amino acid and concomitantly recover a uorescent signal 14 . Alternatively, a non-synonymous mutation in the chromophore of a uorescent protein that induces uorescence spectral change has also been explored as an option to monitor base editing activity 12,15 .…”

“…Alternatively, a non-synonymous mutation in the chromophore of a uorescent protein that induces uorescence spectral change has also been explored as an option to monitor base editing activity 12,15 . Although these assays exploit clever strategies, they are limited to only a few target sequences or exhibit high background signal and/or have relatively low sensitivity 12,13,14,15,16 .…”

Design of smart antibody mimetics with photosensitive switches

“…[20] We reasoned that, upon light-triggered sunbody-mCh interaction, paCBE could restore its cytosineto-thymine editing function. To quickly test this idea, we used a "Gene ON" (GO) luciferase reporter system [21] to monitor the activity of paCBE before and after light stimulation (Figure 6e).…”

A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells

“…Despite the advantages of BEs, the identification of edited cell populations requires the use of end-point sequencing assays. In addition, it has been shown that various parameters including editor expression, nuclease activity, target site accessibility, and sgRNA pairing can limit the efficiency of BE-mediated genome modification [6][7][8][9][10]. In particular, this could limit the use of BEs to modify endogenous target loci that are resistant to editing as it might necessitate the laborious screening of a large number of clonal lines to identify cells with the desired mutation.…”