We have found that having the PAM sites oriented to the outside of the target region (Figure) provides the most robust results. To induce a staggered double-strand break at a target location, the two guide RNAs must target opposite strands.
PAM-out orientation (right side)
(light blue = genomic DNA, dark blue = protospacer, and orange = PAM sequence):
In addition, for optimal results, the distance between cleavage sites should be 40–70 bp apart for the Alt-R Cas9 D10A nickase and 50–70 bp apart for the Alt-R Cas9 H840A nickase. For additional information, refer to the application note at www.idtdna.com/CRISPR-Cas9 (Resources section).