When using the Alt-R® nickases, what orientation should my guides have? Should they target the same or opposite strands?

Aug 16, 2017, 15:06 PM

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):

Pam site orientation (Cas9 nickase experiments)

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 (Support section).

  • Genome Editing
  • Genome Editing
  • Alt-R
  • Cas9