Improved Methods for Deamination-Based m(6)A Detection

N (6)-methyladenosine (m(6)A) is a critical regulator of gene expression and cellular function. Much of our knowledge of m(6)A has been enabled by the identification of m(6)A sites transcriptome-wide. However, global m(6)A profiling methods require high amounts of input RNA to accurately identify methylated RNAs, making m(6)A profiling from rare cell types or scarce tissue samples infeasible. To overcome this issue, we previously developed DART-seq, which relies on the expression of a fusion protein consisting of the APOBEC1 cytidine deaminase tethered to the m(6)A-binding YTH domain. APOBEC1-YTH directs C-to-U mutations adjacent to m(6)A sites, therefore enabling single nucleotide-resolution m(6)A mapping. Here, we present an improved version of DART-seq which utilizes a variant of the YTH domain engineered to achieve enhanced m(6)A recognition. In addition, we develop in vitro DART-seq and show that it performs similarly to cellular DART-seq and can map m(6)A in any sample of interest using nanogram amounts of total RNA. Altogether, these improvements to the DART-seq approach will enable better m(6)A detection and will facilitate the mapping of m(6)A in samples not previously amenable to global m(6)A profiling.