Table 2. Indel mutation frequencies at on- and off-target genomic sites induced by different amounts of Cas9- and sgRNA-expressing plasmids for the RGEN targeted to VEGFA Target Site 2.

Amounts of sgRNA- and Cas9-expressing plasmids transfected into U2OS.EGFP cells for these assays are shown at the top of each column. (Note that data for 250 ng sgRNA/750 ng Cas9 are the same as those presented in Table 1.) Mean indel frequencies were determined using the T7EI assay from replicate samples as described in Online Methods.

Site	Sequence	250ng sgRNA/750 ng Cas9 Mean indel frequency (%) ± SEM	12.5ng sgRNA/50 ng Cas9 Mean indel frequency (%) ± SEM
T2 (On-target)	GACCCCCTCCACCCCGCCTCCGG	50.2 ± 4.9	25.4 ± 4.8
OT2-1	GACCCCCCCCACCCCGCCCCCGG	14.4 ±3.4	4.2 ± 0.2
OT2-2	GGGCCCCTCCACCCCGCCTCTGG	20.0 ± 6.2	9.8 ± 1.1
OT2-6	CTACCCCTCCACCCCGCCTCCGG	8.2 ± 1.4	6.0 ± 0.5
OT2-9	GCCCCCACCCACCCCGCCTCTGG	50.7 ± 5.6	16.4 ± 2.1
OT2-15	TACCCCCCACACCCCGCCTCTGG	9.7 ± 4.5	2.1 ± 0.0
OT2-17	ACACCCCCCCACCCCGCCTCAGG	14.0 ± 2.8	7.1 ± 0.0
OT2-19	ATTCCCCCCCACCCCGCCTCAGG	17.0 ± 3.3	9.2 ± 0.4
OT2-20	CCCCACCCCCACCCCGCCTCAGG	6.1 ± 1.3	N.D.
OT2-23	CGCCCTCCCCACCCCGCCTCCGG	44.4 ± 6.7	35.1 ± 1.8
OT2-24	CTCCCCACCCACCCCGCCTCAGG	62.8 ± 5.0	44.1 ± 4.5
OT2-29	TGCCCCTCCCACCCCGCCTCTGG	13.8 ± 5.2	5.0 ± 0.2
OT2-34	AGGCCCCCACACCCCGCCTCAGG	2.8 ± 1.5	N.D.

OT = Off-target sites, numbered as in Table 1 and Supplementary Table 2. Mismatches from the on-target site (within the 20 bp region to which the gRNA hybridizes) are highlighted as bold, underlined text. N.D. = none detected