FAQ: How should offset sgRNAs be oriented for efficient modification using EnGen® Spy Cas9 Nickase?

Reports on genome modification using Cas9 Nickase (D10A or H840A) suggest efficient modification occurs when sgRNAs are oriented in a tail-to-tail (i.e., 5´ – 5´) orientation and when separated by 10 and 30 bp. Cleavage in this orientation results in 5´ overhanging ends. Editing was detectable with overlapping sgRNA pairs and with pairs at greater distances apart (≥40 bp), albeit with reduced efficiency (Ran et al. (2013) Cell, 155, 479-480). We recommend trying an assortment of sgRNA pairs to identify a set that provides the desired editing efficiency.