Synthetic oligonucleotide-based sequencing of off-target regions for a guide RNA targeting EMX1

To generate a test data to facilitate development of toolkit for off-target nomination data analysis, a library consisting >81K in silico predicted on- and off-target regions for an EMX1 gRNA are synthesized and cleaved in vitro with SpCas9. The cleaved products are used to build NGS library followed by sequencing leading to >13 million reads. About 99% of examined regions are covered by 10 or more valid UMIs. More than 90% of valid UMIs from on-target region support cleavage. About 500 off-target regions have a score greater than 0.1, including 7 off-target regions with score exceeding one. Off-target normination study of a guide RNA targeting EMX1 gene using synthetic oligonucleotide-based sequencing technology