nature methods Transient cold shock enhances zincfinger nuclease mediated gene disruption Yannick Doyon, Vivian M Choi, Danny Xia, Thuy D Vo, Philip D Gregory & Michael C Holmes Supplementary figures and text: Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Supplementary Table 1 Supplementary Table 2 Effect of the cold shock on activity at various ZFN doses. Long term stability of the cells modified using the cold shock method. Effect of the cold shock on ZFN activity and expression levels in HeLa cells. Effect of the cold shock on ZFN activity and expression levels in K562 cells. Preferential cleavage of the target sequence under cold shock conditions. Summary of cell lines tested for ZFN activity under cold shock conditions. Primers used for the Cel1 assays.
Supplementary Figure 1 Effect of the cold shock on activity at various ZFN doses. a TSC2 (K562, day 3) 3 days <1 <1 2 3 18 29 <1 <1 4 7 20 33 <1 1 13 15 31 42 b RIPK1 (K562, day 3) 3 days <1 <1 1 2 1 10 <1 1 6 5 6 28 1 9 18 18 11 35 c KDR (K562, day 3) 3 days * * 1 2 4 4 9 12 2 6 14 8 25 37 7 12 31 26 36 40
(a) K562 cells were nucleofected with a GFP expression plasmid () or 0.1, 0.2 and 0.4 µg of driven ZFN expression vector (lanes 24, 911, and 1618) or a β globin introncontaining vector (lanes 57, 1214, and 1921) encoding ZFN targeting TSC2. Immediately after transfection, cells were divided and incubated for 3 days at 37 C (lanes 17), 1 day at 37 C followed by 2 days at C (lanes 814) or 3 days at C (lanes 1521). The frequency of indels was assessed by the Cel1 assay 3 days posttransfection. (b) Same as (a) but using ZFN targeting RIPK1. (c) Same as (a) but using ZFN targeting KDR. Note that the site targeted is different than the one shown in figure 1 and supplementary figures 3 and 4. * Denotes a cleavage product derived from a SNP. Arrows denote the specific Cel1 product.
Supplementary Figure 2 Long term stability of the cells modified using the cold shock method. a TSC2 (K562, day 10) 3 days <1 <1 1 3 10 20 <1 <1 2 4 15 27 <1 1 11 11 21 47 b RIPK1 (K562, day 10) 3 days <1 <1 1 1 2 11 <1 2 4 4 5 22 1 8 17 15 12 38 c KDR (K562, day 10) 3 days * * <1 1 2 2 5 9 <1 3 8 5 15 32 5 9 20 23 24 29
(a) K562 cells were nucleofected with a GFP expression plasmid () or 0.1, 0.2 and 0.4 µg of driven ZFN expression vector (lanes 24, 911, and 1618) or a β globin introncontaining vector (lanes 57, 1214, and 1921) encoding ZFN targeting TSC2. Immediately after transfection, cells were divided and incubated for 3 days at 37 C (lanes 17), 1 day at 37 C followed by 2 days at C (lanes 814) or 3 days at C (lanes 1521). The frequency of indels was assessed by the Cel1 assay 10 days posttransfection. (b) Same as (a) but using ZFN targeting RIPK1. (c) Same as (a) but using ZFN targeting KDR. Note that the site targeted is different than the one shown in figure 1 and supplementary figures 3 and 4. * Denotes a cleavage product derived from a SNP. Arrows denote the specific Cel1 product.
Supplementary Figure 3 Effect of the cold shock on ZFN activity and expression levels in HeLa cells. a KDR (HeLa, day 3, Cel1) 3 days 1 4 7 6 9 12 8 20 29 37 42 50 40 36 33 28 26 24 b KDR (HeLa, day 10, Cel1) 3 days <1 1 2 2 3 5 11 18 24 21 23 28 35 38 29 24 22 15 c KDR (HeLa, day 3, western) 3 days αnfκb p65 αflag
(a) HeLa cells were nucleofected with a GFP expression plasmid () or 0.1, 0.2 and 0.4 µg of driven ZFN expression vector (lanes 24, 911, and 1618) or a β globin introncontaining vector (lanes 57, 1214, and 1921) encoding KDR ZFNs. Immediately after transfection, cells were divided and incubated for 3 days at 37 C (lanes 17), 1 day at 37 C followed by 2 days at C (lanes 814) or 3 days at C (lanes 15 21). The frequency of indels was assessed by the Cel1 assay 3 days posttransfection. (b) The frequency of indels was assessed by the Cel1 assay 10 days posttransfection. (c) ZFN expression levels were determined by western blot on total cell extracts prepared 3 days posttransfection. The αnfκb and αflag (ZFN) antibodies were coincubated to facilitate normalization between gels.
Supplementary Figure 4 Effect of the cold shock on ZFN activity and expression levels in K562 cells. a KDR (K562, day 3, Cel1) 3 days <1 <1 3 <1 2 5 2 5 17 5 10 31 6 16 35 22 31 43 b KDR (K562, day 10, Cel1) 3 days <1 <1 2 <1 1 3 1 4 17 4 10 27 7 12 23 17 22 27 c KDR (K562, day 3, western) 3 days αnfκb p65 αflag
(a) K562 cells were nucleofected with a GFP expression plasmid () or 0.1, 0.2 and 0.4 µg of driven ZFN expression vector (lanes 24, 911, and 1618) or a β globin introncontaining vector (lanes 57, 1214, and 1921) encoding KDR ZFNs. Immediately after transfection, cells were divided and incubated for 3 days at 37 C (lanes 17), 1 day at 37 C followed by 2 days at C (lanes 814) or 3 days at C (lanes 15 21). The frequency of indels was assessed by the Cel1 assay 3 days posttransfection. (b) The frequency of indels was assessed by the Cel1 assay 10 days posttransfection. (c) ZFN expression levels were determined by western blot on total cell extracts prepared 3 days posttransfection. The αnfκb and αflag (ZFN) antibodies were coincubated to facilitate normalization between gels.
Supplementary Figure 5 Preferential cleavage of the target sequence under cold shock conditions. a Obligate heterodimer 37 + + + + 1 2 3 4 5 Wildtype FokI 37 + + + + ZFN 6 7 8 9 10 GR 7 5 22 19 15 17 40 41 b Obligate heterodimer 37 + + + + 1 2 3 4 5 Wildtype FokI 37 + + + + ZFN 6 7 8 9 10 Trim26 (Intron 1) 2 2 4 3 3 3 24 26 c Obligate heterodimer 37 + + + + 1 2 3 4 5 Wildtype FokI 37 + + + + ZFN 6 7 8 9 10 Chr. 1 (Intergenic) <1 <1 1.4 1.5 <1 <1 17 16
Supplementary Figure 5 Preferential cleavage of the target sequence under cold shock conditions. (a) K562 cells were nucleofected with a GFP expression plasmid () or 80 ng of driven ZFN expression vector (lanes 25, and 710) encoding ZFNs targeting the GR gene. Immediately after transfection, cells were divided and incubated for 3 days at 37 C (lanes 23, and 78), or 3 days at C (lanes 45, and 910). The heterodimeric variants (lanes 25) and the wildtype FokI domain (lanes 710) were compared. The frequency of indels at the GR locus was assessed by the Cel1 assay 3 days posttransfection. (b) The frequency of indels in the intron 1 of Trim26 (offtarget) was assessed by the Cel1 assay 3 days posttransfection. (c) The frequency of indels in an intergenic region of chromosome 1 (offtarget) was assessed by the Cel1 assay 3 days posttransfection.
Supplementary Table 1 Summary of cell lines tested for ZFN activity under cold shock conditions a. Species Cell type # genes targeted # unique sites Fold increase FokI domain Delivery method in Cel1 signal Human K562 15 16 1.5 b 7 c wt, OH d nucleofection Human HeLa 4 4 215 e OH nucleofection Human HEK 293 4 4 1.5 b 15 wt, OH lipofection nucleofection Mouse Splenocytes 1 1 2 wt, OH nucleofection Rat C6 1 1 4 wt lentivirus Hamster CHOK1 3 3 1.5 f 2 b wt, OH nucleofection Pig PK15 1 4 312 OH nucleofection a In all cases, the cold shock treatment was 3 days at C immediately posttransfection. b These ZFNs produce >15% Cel1 signal at 37 C c Undetectable activity at 37 C (< 1% Cel1 signal) d Obligate heterodimeric FokI domain 12 e Cel1 signal saturated at C f These ZFNs produce >20% Cel1 signal at 37 C
Supplementary Table 2 Primers used for the Cel1 assays. AAVS1 KDR (Fig. 1, S3, S4) TP73 MAP3K14 EP0 BTK135 CARM1 GNAI2 TSC2 RIPK1 KDR (Fig. S1,S2) GR TRIM26 Chr. 1 F CCCCTTACCTCTCTAGTCTGTGC R CTCAGGTTCTGGGAGAGGGTAG F AGTATGGGGCCCTGTTGAAT R TCCCACTGACCTTCTATTATGAAA F GGGACAGGACGACTGACTGT R TCAGGGACTAGGGGAACTCA F GAGTATGTGTGGCAGAGGCA R GTGCCCTTGGTAATCACGTC F TTGGAGCACGACTTACCAGA R CCATGGCAGGCTGATTTACT F GAACTCCTGCACTGGTGCTT R GGTGTGGACTCACCCATTTT F GCTAGATGAGGGCTGACTGG R GCCCAAATCTCTGTGAGAGC F AGCTGCATTCCTGGTAGCTC R ACTGGGACCTGCTTCAGAGA F CTGAGAGGGCTGAGGGTGT R AGTGCAGAAACCTGGAAAGC F TGTGGGAAGAGGACCATCTC R GGTAGTTGGCTTCGTCTTGG F GGCTGCGTTGGAAGTTATTT R TGCTAAAAGTCAATGGTATTTTCAA F TCATAACACTGTTCTTCCCCTTCTTTAGCC R TCAAAACACACACTACCTTCCACTGCTC F GGAGTGGTACTGGGCGTGTC R TTCAGGAGGTTTAGAGACCATCAAA F GCAGCAACCTGCCAGCTCTA R CCCATTTGCCAACCAAGAGA