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How do I choose which two guide RNAs to multiplex, when using the Alt-R® nickases?
There are multiple factors to consider when selecting the guide RNAs to multiplex with Cas9 nickase:
Each crRNA should function efficiently in individual genome editing experiments.
The two crRNAs must target opposite strands to ensure a double-strand break is created.
Distance between cleavage sites is important for optimal performance.
Alt-R Cas9 D10A Nickase 3NLS: 40–70 bp
Alt-R Cas9 H840A Nickase 3NLS: 50–70 bp
Outward PAM site orientation (right side)
For more information about the design and use of Cas9 nickases, refer to the application note at www.idtdna.com/CRISPR-Cas9 (Support section).