Frequently asked questions

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

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 (Support section).