Generating, modeling, and evaluating a large-scale set of CRISPR/Cas9 off-target sites with bulges

The CRISPR/Cas9 system is a highly accurate geneediting technique, but it can also lead to unintended off-target sites (OTS). Consequently, many high-throughput assays have been developed to measure OTS in a genome-wide manner, and their data was used to train machine-learning models to predict OTS. However, these models are inaccurate when considering OTS with bulges due to limited data compared to OTS without bulges. Recently, CHANGE-seq, a new in vitro technique to detect OTS, was used to produce a dataset of unprecedented scale and quality. In addition, the same study produced in cellula GUIDE-seq experiments, but none of these experiments included bulges. Here, we generated the most comprehensive GUIDE-seq dataset with bulges, and trained and evaluated state-of-the-art machine-learning models that consider OTS with bulges. We first reprocessed the publicly available experimental raw data of the CHANGE-seq study to gener-ate 20 new GUIDE-seq experiments, and hundreds of OTS with bulges among the original and new GUIDE-seq experiments. We then trained multiple machine-learning models, and demonstrated their state-of-the-art performance both in vitro and in cellula overall and when focusing on OTS with bulges. Last, we visualized the key features learned by our models on OTS with bulges in a unique representation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/565099v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1652c65org.highwire.dtl.DTLVardef@145d719org.highwire.dtl.DTLVardef@2ccdd6org.highwire.dtl.DTLVardef@17dd415_HPS_FORMAT_FIGEXP M_FIG C_FIG