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A Supplemental Fig S1| PLXND1 Genomic Locus. A, Image from the UCSC Genome Browser (Grch37/Hg19). PLXND1 Gene Is Shown in Dark B

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A Supplemental Fig S1| PLXND1 Genomic Locus. A, Image from the UCSC Genome Browser (Grch37/Hg19). PLXND1 Gene Is Shown in Dark B a hg19 20 kb Scale chr3: 129,330,000 129,325,000 129,320,000 129,315,000 129,310,000 129,305,000 129,300,000 129,295,000 129,290,000 129,285,000 129,280,000 129,275,000 P1 P2 P3 P4 P+ Basic Gene Annotation Set from GENCODE Version 24lift37 (Ensembl 83) PLXND1 RN7SL752P PLXND1 10 HeLa-S3 whole cell polyA+ RNA-seq Minus signal Rep 1 from ENCODE/CSHL 1 10 HeLa-S3 whole cell polyA+ RNA-seq Minus signal Rep 2 from ENCODE/CSHL 1 10 HeLa-S3 whole cell polyA+ RNA-seq Plus signal Rep 1 from ENCODE/CSHL 1 10 HeLa-S3 whole cell polyA+ RNA-seq Plus signal Rep 2 from ENCODE/CSHL 1 10 HCT-116 200 bp paired read RNA-seq Signal Rep 1 from ENCODE/Caltech 1 10 HCT-116 200 bp paired read RNA-seq Signal Rep 2 from ENCODE/Caltech 1 Supplemental Fig S1| PLXND1 genomic locus. a, Image from the UCSC Genome Browser (GRCh37/hg19). PLXND1 gene is shown in dark blue (exons are represented as thick boxes and introns as lines). sgRNA pairs are depicted (P1, P2, P3, P4, P+) around exon 1. Whole-cell RNA expression in HeLa (ENCODE/Cold Spring Harbor Lab; plus and minus strand) and HCT116 (ENCODE/Caltech). HeLa (M3814) (n=3) Fraction of plexin D1 negative cells Standard Average deviation Vehicle 0.909 0.013 P+ 300nM 0.896 0.017 P1.1 P1.2 Vehicle 0.013 0.015 P1.1 P2.1 P2.2 300nM 0.000 0.008 Vehicle 0.009 0.005 P3.1 P3.2 P1.2 300nM 0.004 0.007 P4.1 P4.2 400 bp Vehicle 0.004 0.011 P+ P2.1 300nM 0.002 0.009 PLXND1 gene Vehicle 0.006 0.009 chr3:129,325,797 TSS Exon 1 Intron 1 Exon 2 P2.2 300nM 0.003 0.009 Vehicle 0.006 0.012 P3.1 300nM 0.002 0.007 Vehicle 0.001 0.009 P3.2 300nM 0.000 0.008 Vehicle 0.016 0.015 P4.1 300nM 0.012 0.012 Vehicle 0.016 0.014 P4.2 300nM 0.010 0.008 Supplemental Fig S2| Effect of single sgRNAs around PLXND1 first exon. Left: CRISPR-del efficiency of each single sgRNA as measured by CiDER in HeLa with and without DNA-PKcs inhibition (mean, standard deviation). Right: Scheme of CiDER single sgRNAs. a P3 4550 bp PLXND1 gene chr3:129,325,797 (hg19) Non-deletions Inversions b c Non-deleted alleles in HeLa (M3814) Inverted alleles in various cell lines (M3814) (n=3) (n=3) p = 0.009 s e p = 0.134 p = 0.094 s l 1.0 e e l l l p = 0.009 2.0 e p = 0.254 l p = 0.916 a l a d e d t 1.5 e e t l r e e d v - n n 1.0 0.5 i o f n o More deletion f n o More inversion 0.5 o i n t o c i t a r c 0.0 a F r F 0.0 P3 vehicleP3 300nMP3 900nM P3 vehicleP3 300nM P3 vehicleP3 300nM nt vehicle P3 vehicle P3 300nM P3 900nM HeLa HCT116 HEK293T Supplemental Fig S3| . Effect of DNA-PKcs inhibition in the PLXND1 locus. a, Scheme of the locus showing the sgRNA pair used (P3) and QC-PCR primers used to detect non-deleted and inverted alleles. b, CRISPR-del efficiency in HeLa cells. The bar plots show the fraction of non-deleted alleles in the pooled population quantified by qPCR (mean, standard deviation, 1-tailed paired t-test on the 2^dCt values previous to normalization to the vehicle samples). The black bar corresponds to a nontargeting control used for normalization in the ratio calculation. Blue bars correspond to non-treated and M3814-treated samples targeting PLXND1 (P3). c, Proportion of inverted alleles in the pooled population quantified by qPCR and normalized to the P3 non-treated (vehicle) samples (mean, standard deviation, 2-tailed paired t-test on the 2^dCt values previous to normalization to the vehicle samples). a s l HeLa (M3814) l e (n=4) c 0.8 e v i t a g e 0.6 n 1 D n 0.4 i x e l p More deletion f 0.2 o n o i t c 0.0 a r le le M M M M M M M M M F c c n n n n n n n n n i i 1 1 3 0 0 0 0 0 0 h h . 1 3 0 0 0 0 e e 0 1 3 0 0 V V 1 3 P+ P3 M3814 Concentration b s HeLa (M3814) l l e (n=3) c 0.5 e v i t a g 0.4 e n 1 0.3 D n i x e 0.2 l p More deletion f o 0.1 n o i t c 0.0 a r le M le M le M le M le M le M le M le M le M le M F ic n ic n ic n ic n ic n ic n ic n ic n ic n ic n h 0 h 0 h 0 h 0 h 0 h 0 h 0 h 0 h 0 h 0 e 0 e 0 e 0 e 0 e 0 e 0 e 0 e 0 e 0 e 0 V 3 V 3 V 3 V 3 V 3 V 3 V 3 V 3 V 3 V 3 30’ 6h 18h 3d 8d 30’ 6h 18h 3d 8d P1 P3 Dosage time c pDNA-PKcs-469kDa α-Tubulin-50kDa nt nt nt P3 P3 P3 - 300nM 3000nM - 300nM 3000nM tDNA-PKcs-469kDa α-Tubulin-50kDa nt nt nt P3 P3 P3 - 300nM 3000nM - 300nM 3000nM Supplemental Fig S4| Effect of different DNA-PKcs inhibition conditions. a, Dose-response curve performed with by CiDER with pgRNA P3 (green). The positive PLXND1 ORF-targeting control (P+) is shown in tan. b, Effect of different DNA-PKcs inhibition duration on CRISPR-del efficiency (30 minutes, 6 hours, 18 hours, 3 days and uninterrupted inhibition for 8 days). CiDER pgRNAs P1 and P3 were used. The graph displays the fraction of plexin D1 negative cells detected by flow cytometry (mean, standard deviation). c, Detection of active phosphorylated DNA-PKcs (pDNA-PKcs) in untreated (-) and M3814-treated (300nM and 3000nM) samples with pgRNAs targeting PLXND1 (P3) and a nontargeting control (nt). Upper panel: western blot detecting pDNA-PK. α-Tubulin is used as loading control. Bottom panel: total levels of DNA-PKcs (tDNA-PKcs). a Regular genomic PCR for PLXND1-P3 (Biological replicate 1) P3 vehicle P3 M3814 ExpectedExpecte dfull full length lenght (WT) band Non-specificNot desired product band DeletionDeletion band b PLXND1-P3 vehicle sgRNA A cutting site sgRNA B cutting site WT CAATAGCGCTCGCCTCGGGGGAATCTGT---- ----TTTGGTGTAAGCTGATCTGTGGGA 1- CAATAGCGCTCGCCTCG--------------- -------GGTGTAAGCTGATCTGTGGGA 2- CAATAGCGCTCGCCTCG--------------- -------GGTGTAAGCTGATCTGTGGGA 3- CAATAGCGCTCGCCTCGG-------------- -------GGTGTAAGCTGATCTGTGGGA 4- CAATAGCGCTCGCCTCG--------------- -------GGTGTAAGCTGATCTGTGGGA 5- CAATAGCGCTCGCCTCGGG------------- -------GGTGTAAGCTGATCTGTGGGA 6- CAATAGCGCTCGCCTCGG-------------- -------GGTGTAAGCTGATCTGTGGGA Deletion 7- CAATAGCGCTCGCCTCGG-------------- -------GGTGTAAGCTGATCTGTGGGA 8- CAATAGCGCTCGCCTCGGG------------- -------------AGCTGATCTGTGGGA 9- CAATAGCGCTCGCCTCGG-------------- -------GGTGTAAGCTGATCTGTGGGA 10- CAATAGCGCTCGCCTCGG-------------- -------GGTGTAAGCTGATCTGTGGGA 11- CAATAGCGCTCGCCTCGG-------------- -------GGTGTAAGCTGATCTGTGGGA 12- CAATAGCGCTCGCCTCGG-------------- -------GGTGTAAGCTGATCTGTGGGA ***************** *************** PLXND1-P3 M3814 300nM WT CAATAGCGCTCGCCTCGGGGGAATCTGT---- ----TTTGGTGTAAGCTGATCTGTGGGA 1+ CAATAGCGCTCGCCTCGGG------------- -------GGTGTAAGCTGATCTGTGGGA 2+ CAATAGCGCTCGCCTCGG-------------- -------GGTGTAAGCTGATCTGTGGGA 3+ CAATAGCGCTCGCCTCGG-------------- -------GGTGTAAGCTGATCTGTGGGA 4+ CAATAGCGCTCGCCTC---------------- -------GGTGTAAGCTGATCTGTGGGA 5+ CAATAGCGCTC--------------------- --------------GCTGATCTGTGGGA Deletion 6+ CAATAGCGCTCGCCTCGG-------------- -------GGTGTAAGCTGATCTGTGGGA 7+ CAATAGCGCTCGCCTCGG-------------- -------GGTGTAAGCTGATCTGTGGGA 8+ CAATAGCGCTCGCCTC---------------- -------GGTGTAAGCTGATCTGTGGGA 9+ CAATAGCGCTCGCCTCGG-------------- -------GGTGTAAGCTGATCTGTGGGA *********** ************** Supplemental Fig S5| CiDER deletion outcomes in DNA-PKcs inhibition untreated and treated cells. a, Genomic PCR amplification of the PLXND1 gene from untreated and treated CiDER samples using the pgRNA-P3. Deletion bands are boxed. b, Sanger sequencing alignment results of TOPO cloned alleles of purified PCR deletion bands from (a). Expected sgRNAs cut sites are indicated by vertical lines. a b HCT116 (M3814) HEK293T (M3814) (n=4) (n=4) p=0.024 ns ns s s e e l 1.0 ns l 1.0 ns e p=0.003 e l l p=0.020 P5 1500 bp l l a a ns 0.8 0.8 P6 1600 bp P7 1650 bp 0.6 0.6 1400 bp P8 non-deleted non-deleted 0.4 0.4 f f o o n n o 0.2 o 0.2 i i More deletion chr11:65,254,282 (hg19) QC-PCR t t c c a a r r F 0.0 F 0.0 le M le M le M le M le M le M le M le M ic n ic n ic n ic n ic n ic n ic n ic n h 0 h 0 h 0 h 0 h 0 h 0 h 0 h 0 e 0 e 0 e 0 e 0 e 0 e 0 e 0 e 0 V 3 V 3 V 3 V 3 V 3 V 3 V 3 V 3 P5 P6 P7 P8 P5 P6 P7 P8 Supplemental Fig S6| Effect of DNA-PKcs inhibition on deletion of the MALAT1 enhancer locus. a, Scheme of the sgRNA pairs and QC-PCR primers. b, CRISPR-del efficiency in HCT116 and HEK293T upon DNA-PK inhibition. The bar plots show the fraction of non-deleted alleles in the pooled population quantified by qPCR (mean, standard deviation, 1-tailed paired t-test). a Genomic PCR for MALAT1-enhancer-P5 (Biological replicate 1) P5 P5 Expected full length band Deletion band b MALAT1-enhancer-P5 vehicle sgRNA A cutting site sgRNA B cutting site WT TATAGTTCTGCCTCAGCTCAGGACG---- ----GTCTTTACTTGACCAACACTGA 1- TATAGTTCTGCCTCAGCTCA--------- ----GTCTTTACTTGACCAACACTGA 2- TATAGTTCTGCCTCAGCTCA--------- ----GTCTTTACTTGACCAACACTGA 3- TATAGTTCTGCCTCAGCTC---------- ----GTCTTTACTTGACCAACACTGA 4- TATAGTTCTGCCTCAGCTCA--------- ----GTCTTTACTTGACCAACACTGA 5- TATAGTTCTGCCTCAGCTCA--------- ----GTCTTTACTTGACCAACACTGA 6- TATAGTTCTGCCTCAGCTC---------- ----GTCTTTACTTGACCAACACTGA Deletion 7- TATAGTTCTGCCTCAGCTC---------- ----GTCTTTACTTGACCAACACTGA 8- TATAGTTCTGCCTCAGCTC---------- ----GTCTTTACTTGACCAACACTGA 9- TATAGTTCTGCCTCAGCTCA--------- ----GTCTTTACTTGACCAACACTGA 10- TATAGTTCTGCCTCAGCTCA--------- ----GTCTTTACTTGACCAACACTGA 11- TATAGTTCTGCCTCAG------------- -----------CTTGACCAACACTGA **************** *************** MALAT1-enhancer-P5 M3814 300nM WT TATAGTTCTGCCTCAGCTCAGGACG---- ----GTCTTTACTTGACCAACACTGA 1+ TATAGT----------------------- -----TCTTTACTTGACCAACACTGA 2+ TATAGTTCTGCCTCAGCTCA--------- ----GTCTTTACTTGACCAACACTGA 3+ TATAGTTCTGCCTCAGC------------ -----TCTTTACTTGACCAACACTGA 4+ TATAGTTCTGCCTCAGCTC---------- ----GTCTTTACTTGACCAACACTGA 5+ TATAGTTCTGCCTCAGCTCA--------- ----GTCTTTACTTGACCAACACTGA Deletion 6+ TATAGTTCTGCCTCAGCTCA--------- ----GTCTTTACTTGACCAACACTGA 7+ TATAGTTCTGCCTCAGCTCA--------- -----TCTTTACTTGACCAACACTGA 8+ TATAGTTCTGCCTCAGCTCA--------- ----GTCTTTACTTGACCAACACTGA 9+ TATAGTTCTGCCTCAGCTCA--------- ----GTCTTTACTTGACCAACACTGA 10+ TATAGTTCTGCCTCAGCT----------- -----------------CAACACTGA 11+ TATAGTTCTGCCTCAGCTCA--------- ----GTCTTTACTTGACCAACACTGA ****** ********* *Figure legend in the next page.
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