Table 2. DNA template design for off-target binding sites.

PAM Site	Template ID	DNA Sequence
PAM1	Native	GAGAAAATAATGAATGTCAA
PAM1	Off-Target #1	GAGAAAATAATTAATGTAAG
PAM1	Off-Target #2	GAGAAAATAATGAATGTTCA
PAM2	Native	AATGAATGTCAAAGGAAGAG
PAM2	Off-Target #1	AATGAATGTCAATGGAAGTG
PAM2	Off-Target #2	AATCAATGTCAAATGAAGAG
PAM3	Native	GCTGCTTGTCTCAACTGTAA
PAM3	Off-Target #1	GCTGCTTGTCTCAACTATAA
PAM3	Off-Target #2	GCTGCTTGGCTCTACTGTAA

The native sequence remained constant across all PAM sites and the unmutated region of each PAM site is shown for reference. Mutated bases within the 20 bp spacer region preceding the PAM site are shown bolded and underlined.