CHANGE-seq-BE enables simultaneously sensitive and unbiasedin vitroprofiling of base editor genome-wide activity

Abstract

Base editors (BE) enable programmable conversion of nucleotides in genomic DNA without double-stranded breaks and have substantial promise to become new transformative genome editing medicines. Sensitive and unbiased detection of base editor off-target effects is important for identifying safety risks unique to base editors and translation to human therapeutics, as well as accurate use in life sciences research. However, current methods for understanding the global activities of base editors have limitations in terms of sensitivity or bias. Here we present CHANGE-seq-BE, a novel method to directly assess the off-target profile of base editors that is simultaneously sensitive and unbiased. CHANGE-seq-BE is based on the principle of selective sequencing of adenine base editor modified genomic DNA in vitro, and provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of base editors.

Selective haematological cancer eradication with preserved haematopoiesis

“…To nominate potential off-target editing sites we performed CHANGE-seq-BE 28 on genomic DNA from the same donor used in this engraftment experiment (Extended Data Fig. 6f).…”

“…Genomic DNA was extracted from human peripheral blood mononuclear cells (PBMCs) using the Puregene tissue kit (Qiagen, 158063) according to the manufacturer's instructions including the proteinase-K and RNase steps (Qiagen, 158143 and 158153). CHANGE-seq-BE was adapted and modified from the original CHANGE-seq method 54 to validate genome-wide activity for ABEs 28 . Similar to CHANGE-seq, purified genomic DNA tagmented with a custom Tn5-transposome to generate an average length of 650 bp and followed by gap repair with Kapa HiFi HotStart Uracil + DNA Polymerase (KAPA Biosystems, KK2802) and Taq DNA ligase (NEB, M0208L).…”