. Author manuscript; available in PMC: 2012 Mar 1.

Published in final edited form as: Nat Methods. 2011 Aug 7;8(9):765–770. doi: 10.1038/nmeth.1670

Table 1. CCR5-224 off-target sites in the genome of human K562 cells.

Lower case letters indicate mutations compared to the target site. Sites marked with an ‘X’ were found in the corresponding in vitro selection dataset. ‘T’ refers to the total number of mutations in the site, and ‘(+)’ and ‘(−)’ to the number of mutations in the (+) and (−) half-sites, respectively. The sequences of the sites are listed as 5′ (+) half-site/spacer/(−) half-site 3′, therefore the (+) half-site is listed in the reverse sense as it is in the sequence profiles. K562 modification frequency is the frequency of observed sequences showing significant evidence of non-homologous end joining repair (see Online Methods) in cells expressing active ZFN compared to cells expressing empty vector. Sites that did not show statistically significant evidence of modifications are listed as not detected (n.d.), and K562 modification frequency is left blank for the three sites that were not analyzed due to non-specific PCR amplification from the genome. Supplementary Table S3 shows the sequence counts and P-values for the tested sites used to determine K562 modification frequency, and Supplementary Table S5 shows the modified sequences obtained for each site

mutations			gene				in vitro selection stringency (nM)				K562 modification frequency
T	(+)	(−)	gene	(+) half-site	spacer	(−) half-site	4	2	1	0.5	K562 modification frequency
0	0	0	CCR5 (coding)	GTCATCCTCATC	CTGAT	AAACTGCAAAAG	X	X	X	X	1: 2.3
2	1	1	CCR2 (coding)	GTCgTCCTCATC	TTAAT	AAACTGCAAAAa	X	X	X	X	1: 10
3	2	1	BTBD10 (promoter)	GTttTCCTCATC	AAAGC	AAACTGCAAAAt	X	X			1: 1,400
4	0	4		GTCATCCTCATC	AGAGA	AAACTGgctAAt	X	X			n.d.
4	3	1	SLC4A8	taaATCCTCATC	TCTATA	AAAaTGCAAAAG	X	X			n.d.
3	2	1	Z83955 RNA	GTCATCCcaATC	GAAGAA	AAACTGaAAAAG	X			X	n.d.
3	1	2	DGKK	cTCATCCTCATC	CATGC	AcAaTGCAAAAG	X				n.d.
3	1	2	GALNT13	GTCATCCTCAgC	ATGGG	AAACaGCAgAAG	X				n.d.
3	1	2		GTCATCtTCATC	AAAAG	gAACTGCAAAAc	X				1: 2,800
4	0	4		GTCATCCTCATC	CAATA	AAAgaaCAAAgG	X				n.d.
4	1	3	TACR3	GTCATCtTCATC	AGCAT	AAACTGtAAAgt	X				1: 300
4	1	3	PIWIL2	GTCATCCTCATa	CATAA	AAACTGCcttAG	X
4	1	3		aTCATCCTCATC	CATCC	AAtgTtCAAAAG	X				n.d.
4	3	1		GTCcTgCTCAgC	AAAAG	AAACTGaAAAAG	X				1: 4,000
4	3	1	KCNB2	aTgtTCCTCATC	TCCCG	AAACTGCAAAtG	X				1: 1,400
4	3	1		GTCtTCCTgATg	CTACC	AAACTGgAAAAG	X				1: 5,300
4	3	1		aaCATCCaCATC	ATGAA	AAACTGCAAAAa	X				n.d.
6	3	3		aTCtTCCTCATt	ACAGG	AAAaTGtAAtAG	X				n.d.
6	4	2	CUBN	GgCtTCCTgAcC	CACGG	AAACTGtAAAtG	X
6	5	1	NID1	GTttTgCaCATt	TCAAT	tAACTGCAAAAG	X				n.d.
3	2	1		GTCAaCCTCAaC	ACCTAC	AgACTGCAAAAG	X				1: 1,700
4	1	3	WWOX	GTCATCCTCcTC	CAACTC	cAAtTGCtAAAG	X				n.d.
4	2	2	AMBRA1	GTCtTCCTCcTC	TGCACA	tcACTGCAAAAG	X				n.d.
4	2	2		GTgATaCTCATC	ATCAGC	AAtCTGCAtAAG	X				n.d.
4	2	2	WBSCR17	GTtATCCTCAgC	AAACTA	AAACTGgAAcAG	X				1: 860
4	2	2	ITSN	cTCATgCTCATC	ATTTGT	tAACTGCAAAAt	X				n.d.
4	4	0		GcCAgtCTCAgC	ATGGTG	AAACTGCAAAAG	X				n.d.
4	4	0		cTCATtCTgtTC	ATGAAA	AAACTGCAAAAG	X				n.d.
5	3	2		GaagTCCTCATC	CCGAAG	AAACTGaAAgAG	X				n.d.
5	3	2	ZNF462	GTCtTCCTCtTt	CACATA	AAACcGCAAAtG	X				n.d.
5	4	1		aTaATCCTttTC	TGTTTA	AAACaGCAAAAG	X				n.d.
5	4	1		GaCATCCaaATt	ACATGG	AAACTGaAAAAG	X				n.d.
5	5	0	SDK1	GTCtTgCTgtTg	CACCTC	AAACTGCAAAAG	X				n.d.
4	1	3	SPTB(coding)	GTCATCCgCATC	GCCCTG	gAACTGgAAAAa		X			n.d.
4	2	2		aTCATCCTCAaC	AAACTA	AAACaGgAAAAG		X
4	4	0	KIAA1680	GgaATgCcCATC	ACCACA	AAACTGCAAAAG		X			n.d.
5	5	0		GTttTgCTCcTg	TACTTC	AAACTGCAAAAG		X			n.d.