Supplementary information A combinational CRISPR/Cas9 gene-editing approach can halt HIV replication and prevent viral escape Robert Jan Lebbink, Dorien CM de Jong, Femke Wolters, Elisabeth M Kruse, Petra M van Ham, Emmanuel JHJ Wiertz and Monique Nijhuis* Department of Medical Microbiology, Virology, University Medical Center Utrecht, Utrecht, The Netherlands Supplementary Table S1. gRNA sequences used in this study. Supplementary Table S2. Primer sequences for on-and off-target analysis. Supplementary Table S3. Analysis of off-target activity of anti-HIV gRNAs. Supplementary Table S4. Deep sequence analysis of gRNA target sites after (long-term) in vitro selection experiments. Supplementary Table S5. Nucleotide substitution patterns observed in HIV escape variants. Supplementary Figure S1. Impact of Cas9/gRNA expression on cell growth and viability. SupT1 cells were stably transduced with the single gRNAs. After four days, cell growth and viability was measured in an MTT based cell viability assay. Actively respiring cells convert the watersoluble MTT to an insoluble purple formazan which can be solubilized and its concentration can be measured at a wavelength of 540 nm. Data is presented as a mean with standard deviation from three or more independent assays.Table S1. Selected gRNA sequences used in this study CRISPR ID target gene gRNA sequence PAM GFP1 green fluorescent protein TGAACCGCATCGAGCTGAAG GGG LTR4 HIV-LTR GCCACTCCCCAGTCCCGCCC AGG LTR6 HIV-LTR GCTCAGATCTGGTCTAACCA GAG MA3 HIV matrix (p17) GCGGGGGAGAATTAGATCGA TGG PR1 HIV protease GATGGAAACCAAAAATGATA GGG PR2 HIV protease GCTATAGGTACAGTATTAGT AGG PR3 HIV protease GTATCTAATAGAGCTTCCTT TAG PR4 HIV protease GCAACTAAAGGAAGCTCTAT TAG PR5 HIV protease GGAAGCTCTATTAGATACAG GAG RT2 HIV reverse transcriptase GTACAGAGATGGAAAAGGAA GGG RT3 HIV reverse transcriptase GGAGTATTGTATGGATTTTC AGG RT4 HIV reverse transcriptase GACTTCTGGGAAGTTCAATT AGG RT6 HIV reverse transcriptase GTCTGGATTTTGTTTTCTAA AAG IN1 HIV integrase GATATTTCTCATGTTCATCT TGG IN2 HIV integrase GGACAAGTAGACTGTAGTCC AGG IN4 HIV integrase GTTTCCTGCCCTGTTTCTGC TGG IN5 HIV integrase GGCGGCCTTAACCGTAGCAC TGG IN7 HIV integrase GCCGGTAAAAACAATACATA CAGTable S2. Primer sequences for on- and off-target analysis On - off target sequence mismatches + location Chr. position Fw primer (5'-3') Rev primer (5'-3') LTR6 ON GCTCAGATCTGGTCTAACCAGAG AGAACTGCTGAcATCGAGCTTGC CTTCAGCAAGCCGAGTCCTG LTR6 OFF1 GCCCAGAGCCGGTCTAACCAGAG 3MMs [3:8:10] 20 -19351498 TGCCAACCTATCCCAGCAGG AGACCCACACCAAGACAGGC LTR6 OFF2 TCCAAGAACTGGTCTAACCAAGG 4MMs [1:3:4:8] 4 3277031 ACCCTTTCACAACCACCAGC AGCCCTGACTTCATTGTGCAAC LTR6 OFF3 GTGCAGACCTTGTCTAACCAAGG 4MMs [2:3:8:11] 9 -112431084 TTTGGTCTGTGGGCAAACTG CCAGTGCTAAGCAGACTTGAGG MA3 ON GCGGGGGAGAATTAGATCGATGG CTCTCTCGACGCAGGACTCG TGTGTCCTGTGTCAGCTGCT MA3 OFF1 TCCAGGGAGAATCAGATCGATGG 4MMs [1:3:4:13] 3 -193038771 TCTTATACATCTGGCTGCCGC ACCACAGTGAGGCATCTGGG MA3 OFF2 CCTGTGGAGAATTAGATCTAAGG 4MMs [1:3:5:19] 4 76175203 CTGGCACACTGACAAAGCAG GAAAAAGGCACCCTGTCTGG MA3 OFF3 GAGGGGGAGAATGAGAACGATGG 3MMs [2:13:17] X 40589720 GCCAGCAGACCGGAGAGATG TAGGGGAAAGGGGAGGCAGG IN5 ON GGCGGCCTTAACCGTAGCACTGG GCCAGTGGATATATAGAAGC CCAATCCCCCCTTTTCTTT IN5 OFF1 GGAGGACGTAACTGTAGCACTAG 4MMs [3:6:8:13] 18 -75412696 CTGCTCAGACTGTGCCATCC TTCCTCAGAGGTTATTCTTTCAATG IN5 OFF2 GGCAGCCTGCACTGTAGCACTAG 4MMs [4:9:10:13] 10 119015953 CCCAGTGGTAGGAGGCTGC AGGGTCTTGGCCAGGGTTAC IN5 OFF3 TACTGCCTTAACCGTGGCACAAG 4MMs [1:2:4:16] 7 93632950 GCCGTTCTAGTAGCTGTGCG TGTTTCCCTACTGGCCCTGGTable S3. Analysis of off-target activity of anti-HIV gRNAs gRNA positive cells gRNA negative cells Sequences Sequences gRNA Total Mutant (%) Wildtype (%) Total Mutant (%) Wildtype (%) gRNA pos – gRNA neg (%) LTR6 Off1 2491 0.60 99.40 1241 1.37 98.63 -0.77 Off2 4177 1.01 98.99 1166 0.86 99.14 0.15 Off3 2426 0.91 99.09 951 1.05 98.95 -0.14 MA3 Off1 2259 1.73 98.27 1111 0.81 99.19 0.92 Off2 2007 0.90 99.10 977 0.34 99.66 0.56 Off3 1841 0.43 99.57 770 1.17 98.83 -0.74 IN5 Off1 1743 1.09 98.91 1019 0.59 99.41 0.50 Off2 2487 0.88 99.12 1087 0.65 99.35 0.23 Off3 2903 0.76 99.24 1374 0.66 99.34 0.10Table S4. Deep sequence analysis of gRNA target sites after (long-term) in vitro selection experiments PR RT IN MA gRNA exp # total count seq % total count seq % total count seq % total count seq % control cells 2 864 ATTAGT 99,8 864 AAGGAA 98,8 904 GTGCTA 100,0 235 GATCGA 100,0 empty vector 2 748 ATTAGT 99,7 748 AAGGAA 99,7 873 GTGCTA 100,0 239 GATCGA 100,0 PR2 1 565 ATTAGT 2,5 TTTAGT 69,2 CCTAGT 21,1 22 del 3,9 PR2 2 829 ATTAGT 1,3 GTTAGT 70,3 PR2 3 806 ATTAGT 4,4 TCTAGT 39,3 ATTGAT 39,1 ATTGAAGT 5,1 PR2 4 776 ATTAGT 4,5 ATTGTT 85,8 RT2 1 1532 AAGGAA 50,8 AAG-AA 12,7 ACCGAA 4,4 RT2 2 914 AAGGAA 19,2 ACCGAA 34,4 AAG-AA 8,9 AACGAA 4,1 RT2 3 1063 AAGGAA 37,2 AAG-AA 8,8 AAGGA- 7,3 RT2 4 1268 AAGGAA 32,4 AAG-AA 12,2 ACCGAA 6,4 IN5 1 837 GTGCTA 5,3 GTGCGG 66,9 GTGC-G 12,0 IN5 2 572 GTGCTA 20,8 GCTCTA 27,2 GTGTCA 10,2 GTGGGG 6,0 MA3 1 127 GATCGA 0,0 GACCGA 40,5 GATTTA 34,1 GAGCGA 14,3MA3 2 149 GATCGA 0,0 GACCGA 58,1 GAGCGA 29,1 GA---- 12,8 MA3 3 311 GATCGA 2,9 GACCGA 50,0 GGACGA 10,3 GTCACA 7,4 MA3 4 442 GATCGA 2,3 GAGCGA 66,4 GACCGA 20,6 GGACGA 4,1 RT2 + PR2 1 932 ATTAGT 15,7 932 AAGGAA 1,0 ATTGAC 28,5 GGGGAA 54,8 ACTAGT 11,6 -GGGAA 19,6 ATCAGT 3,0 GTCGAA 2,7 RT2 + IN5 1 775 AAGGAA 2,5 894 GTGCTA 0,6 AACGAA 95,5 GTGAAG 95,6 RT2 + IN5 2 502 AAGGAA 41,3 686 GTGCTA 0,7 AAGGCT 27,4 GTGGTA 50,2 AAG-AA 9,8 GCTCTA 26,6 AAGGA- 6,8 GTAACTA 10,1 RT2 + IN5 3 530 AAGGAA 2,5 691 GTGCTA 2,8 ACAGAA 38,6 GTGGTA 84,9 ACCGAA 31,4 GTG-TA 2,5 ATTGAA 10,4 RT2 + IN5 4 629 AAGGAA 30,3 501 GTGCTA 5,4 AAG-AA 15,6 GTGGAG 66,4 AGCGAA 5,3 GTGGCA 9,6 RT2 + MA3 1 777 AAGGAA 1,3 634 GATCGA 1,9 AGGGAA 66,2 GACCGA 97,9 --GGAA 19,1 A-GGAA 8,8 RT2 + MA3 2 1296 AAGGAA 0,9 432 GATCGA 0,0 ACCGAA 97,0 GACCGA 95,8 GGGCGA 2,3 RT2 + MA3 3 1441 AAGGAA 64,1 645 GATCGA 2,8 AAG-AA 12,2 GAGCGA 87,3 -AGGAA 1,8 GACCGA 4,3 RT2 + MA3 4 506 AAGGAA 39,1 363 GATCGA 0,0 AAG-AA 10,5 GACCGA 88,2 GGGGAA 5,9 GGTCGA 3,3 ATCGAA 4,7Table S5. Nucleotide substitution patterns in HIV escape variants HIV target gene PR RT IN MA Total Samples (n) 14 21 14 18 67 target sequence ATTAGT AAGGAA GTGCTA GATCGA A-->T 4 5 0 1 10 A-->C 2 13 0 0 15 A-->G 10 10 7 10 37 T-->A 0 0* 5 5 10 T-->C 13 0* 5 13 31 T-->G 1 0* 5 18 24 C-->A 0* 0* 1 1 2 C-->T 0* 0* 1 1 2 C-->G 0* 0* 11 0 11 G-->A 2 1 1 1 5 G-->T 1 2 3 1 7 G-->C 1 15 1 1 18 34 46 40 52 172 * Nucleotide is not present in target siteFigure S1. Impact of Cas9/gRNA expression on cell growth and viability 0 20 40 60 80 100 120 140 Cell viability (%)