The CRISPR-Cas9 crRNA recognizes and binds 19–20 nt on the genomic DNA strand opposite to the NGG sequence of the PAM site. We recommend using 20 nt upstream of the PAM site in the forward orientation as your crRNA protospacer sequence.
Also, make sure you verify the correct strand orientation of the protospacer sequence and do not include the PAM site.
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