YMTHE, Volume 25

Supplemental Information

Using CRISPR-Cas9 to Generate Gene-Corrected

Autologous iPSCs for the Treatment of Inherited

Retinal Degeneration

Erin R. Burnight, Manav Gupta, Luke A. Wiley, Kristin R. Anfinson, AudreyTran, Robinson Triboulet, Jeremy M. Hoffmann, Darcey L. Klaahsen, Jeaneen L.Andorf, Chunhua Jiao, Elliott H. Sohn, Malavika K. Adur, Jason W. Ross, Robert F.Mullins, George Q. Daley, Thorsten M. Schlaeger, Edwin M. Stone, and Budd A. Tucker

SUPPLEMENTARY MATERIALS Supplementary Materials and Methods

Design and cloning of MAK CRISPR-Cas9 constructs:

Bicistronic constructs were generated that encoded 1) a chimeric small guide and

transactivating RNA (sgRNA) transcript under the control of the human Pol III U6 promoter and

2) a human codon-optimized S. pyogenes Cas9 nuclease transcript driven by a hybrid chicken

beta-actin (Cbh) promoter (pX330-U6-Chimeric_BB-Cbh-hSpCas9) 1. pX330-U6-Chimeric_BB-

Cbh-hSpCas9 was a gift from Feng Zhang (Addgene plasmid #42230). SgRNAs were designed

using the Optimized CRISPR Design Tool (crispr.mit.edu). Oligonucleotides were synthesized

(Integrated DNA Technologies, Coralville, IA), annealed, phosphorylated and cloned into BbsI

sites directly downstream of the Pol III U6 promoter in pX330-U6-Chimeric_BB-Cbh-hSpCas9

(Table 1).

Cloning of MAK HDR Donor construct:

A donor repair construct for correcting the Alu insertion mutation in MAK was synthesized by

Integrated DNA Technologies (Coralville, IA). The donor plasmid carries the corrected MAK

exon 9 sequence and a floxed puromycin-selection cassette flanked by 500 base pairs

homologous sequence (Figure 1E).

Design and cloning of IVS26 constructs:

Streptomyces pyogenes reagents:

For correcting the CEP290 splicing defect (IVS26 mutation) in patient 2 hiPSCs, single-guide

(sgRNA) plasmids were constructed in which the human U6 promoter drives expression of

chimeric RNA (crRNA/tracrRNA fusion; cassette equivalent to Addgene plasmid # 41817) in a

pUC57 vector backbone (plasmids synthesized by GenScript®). A human codon-optimized S.

pyogenes Cas9-2A-GFP nuclease transcript, driven by CAG promoter, was expressed from a

separate plasmid (pCas9_GFP, a gift from Kiran Musunuru, Addgene plasmid # 44719).

Staphylococcus aureus reagents:

To correct the IVS26 mutation in patients 3 and 4 hiPSCs, an AAV transgene plasmid was

generated that consisted of AAV2 inverted terminal repeats (ITRs) flanking two human U6

promoter-driven sgRNA cassettes downstream of a Cbh-driven humanized S. aureus Cas9

nuclease expression cassette. A KpnI-flanked hU6-BbsI-sgRNA cassette was synthesized

(Integrated DNA Technologies, Coralville, IA) and cloned into a KpnI-linearized pX601-AAV-

CMV::NLS-SaCas9-NLS-3xHA-bGHpA; U6::BsaI-sgRNA plasmid 2. The pX601 plasmid was a

gift from Feng Zhang (Addgene plasmid #61591). The sgRNAs were designed using the

Benchling platform (benchling.com). Oligonucleotides were synthesized (Integrated DNA

Technologies, Coralville, IA), annealed, phosphorylated, and cloned into the BbsI or BsaI sites

in the pX601 backbone (Table 1).

Cloning of IVS26 HDR Donor construct:

A donor repair construct for correcting the IVS26 mutation in CEP290 was synthesized by

GenScript (Piscataway, NJ). The donor plasmid carries the corrected CEP290 exon X sequence

and a floxed puromycin-selection cassette flanked by 500 base pairs homologous sequence.

Design and cloning of RHO P23H constructs:

Staphylococcus aureus reagents:

AAV transgene plasmids were generated that encoded sgRNAs targeting the RHO P23H

mutation and the humanized S. aureus Cas9 nuclease. The sgRNAs were designed using the

Benchling platform (benchling.com). Oligonucleotidess were synthesized (Integrated DNA

Technologies, Coralville, IA), annealed, phosphorylated, and cloned into the BsaI site in the

pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA; U6::BsaI-sgRNA plasmid 2 (Table S1). The

pX601 plasmid was a gift from Feng Zhang (Addgene plasmid #61591).

Streptomyces pyogenes reagents:

Bicistronic constructs were generated that encoded 1) a chimeric small guide and

transactivating RNA (sgRNA) transcript under the control of the human Pol III U6 promoter and

2) a human codon-optimized S. pyogenes Cas9 nuclease transcript driven by a hybrid chicken

beta-actin (Cbh) promoter (pX330-U6-Chimeric_BB-Cbh-hSpCas9) 1. pX330-U6-Chimeric_BB-

Cbh-hSpCas9 was a gift from Feng Zhang (Addgene plasmid #42230). SgRNAs were designed

using the Optimized CRISPR Design Tool (crispr.mit.edu). Oligonucleotides were synthesized

(Integrated DNA Technologies, Coralville, IA), annealed, phosphorylated and cloned into BbsI

sites directly downstream of the Pol III U6 promoter in pX330-U6-Chimeric_BB-Cbh-hSpCas9

(Table 1).

Cloning of P23H HDR Donor construct:

A donor repair construct for correcting the Pro23His mutation in RHO was synthesized by

GenScript (Piscataway, NJ). The donor plasmid carries the corrected RHO exon 1 sequence

and a floxed puromycin-selection cassette flanked by 500 base pairs homologous sequence.

Rt-PCR characterization following CRISPR-mediated correction:

Patient 2 (Figure 2):

For the CEP290 S. pyogenes data presented in Figure 2, one μg of RNA was reverse

transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied

Biosystems). All PCR reactions were performed in a 20μL reaction containing 1x GoTaq®

Green master mix, ~100 ng of cDNA, and 10 µM of each gene specific forward and reverse

primer (Integrated DNA Technologies). All cycling profiles incorporated an initial denaturation

temperature of 95°C for 2 min through 30-35 amplification cycles (45 sec at 95°C, 45 sec at

annealing temperature of each primer and 1 min at 72°C) and a final extension at 72°C for 5

min. PCR products were separated by gel electrophoresis using 2% agarose gels or sent to

Genewiz for Sanger sequencing using the specified primers (Table S1).

Patients 3 and 4 (Figure 3):

Total RNA was isolated using RNeasy Mini-kit (Qiagen, Valencia, CA) according to the

manufacturer’s instructions. 1ug of RNA was reverse transcribed into cDNA using the random

hexamer (Invitrogen, Carlsbad, CA) priming method. All PCR reactions were performed in a 50

uL reaction containing 1x PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 100 ng of DNA, 1.0 U of

Platinum Taq (Invitrogen) and 20 pmol of each gene specific primer (Integrated DNA

Technologies, Coralville, IA). All cycling profiles incorporated an initial denaturation temperature

of 94°C for 10 min through 35 amplification cycles (30 sec at 94°C, 30 sec at annealing

temperature of each primer and 1 min at 68°C) and a final extension at 68°C for 5 min. PCR

products were separated by gel electrophoresis using 2% agarose e-gels (Invitrogen). Primers

used are listed in Table S1.

Western Blot:

Whole cell lysates were made by lysing cells directly in 6-well culture dishes. Lysates were

cleared by centrifugation, and protein concentrations quantified with the Bio-Rad Protein Assay

Dye Reagent Concentrate (BioRad). Thirty μg of protein extract per sample were heat-

denatured in the presence of 2-ME, separated on a 4% Tris-HCl gel (Thermo Fisher Scientific,

Waltham, MA), and transferred to an Immobilon Membrane (Millipore). After blocking with 5%

milk in Tris-Buffered Saline and Tween 20 (TBST), the membrane was incubated with primary

antibody overnight at 4°C (Abcam ab85728; 1:2000). After washing, the secondary antibody

(anti-rabbit-HRP, Santa Cruz sc-2313; 1:20,000) was incubated for 1 hour at room temperature.

After final washes, the antibody signal was visualized using the chemiluminescent Pierce ECL

Western Blotting Substrate (PerkinElmer) and exposure onto Denville Scientific film.

Off-target analysis:

Patients 1 and 3-5: Guides exhibiting the most efficient on-target cutting were subjected to off-

target analysis prior to being used for experiments in patient specific iPSC lines. The fifty most

likely off-target sites were determined using the Optimized CRISPR Design Tool (MAK (Table

S2)) or Benchling platform (IVS26 and P23H; Table S2). All predicted off-target sites that fell

within protein coding regions regardless of rank, and the top 20 predicted off target sites

regardless of genomic location, were evaluated for off-target cutting. Genomic DNA from

sgRNA-treated and untreated control human cells was PCR-amplified and subjected to T7E1

nuclease assays as described above. For the majority of loci, HEK293 cells were used as

controls. However, for the five loci that would not amplify from HEK293 cells (presumably due

to the many genomic rearrangements in these cells 3) HeLa cells were used instead. Off-target

cutting events in protein coding sequences were considered detrimental and warranted testing

of the guide determined to be the next most efficient for directing on-target modification. Patient

2: We assessed the top five off-target loci with the highest overall scores as well as the five

highest-scoring off-target sites with perfect PAM sequence, for each sgRNA (Table S3).

Genomic DNA from sgRNA treated and untreated human iPSCs was PCR amplified for each of

the off-target loci, and analyzed by electrophoresis and sequencing.

Transfection of HEK293Ts and patient-specific iPSCs with MAK CRISPR-Cas9 HDR machinery:

To test HDR using our MAK CRISPR/Cas9 constructs, HEK293T cells were transfected via

lipofection at 1:0, 1:4, 1:2, 2:1, and 4:1 ratios (donor construct: sg1MAK construct) and cultured

in the presence of puromycin (0.5ug/ml) for two weeks. Polyclonal genomic DNA was isolated

and screened via PCR using primers specific to the MAK HDR cassette and MAK sequence

lying outside of the homology arms. 1:4 or 4:1 (donor construct:sg1MAK) ratios of MAK

CRISPR-Cas9 plasmids were delivered to 1.2 x 106 patient-specific iPSCs using Nucleofector™

Technology (Lonza Inc., Mapleton, IL) and pulse code CB130, and then cultured under

puromycin selection (1ug/ml) for two weeks.

Generation of patient-specific induced pluripotent stem cell lines:

Patients 1 and 3-5:

250,000 patient-specific dermal fibroblasts isolated from upper arm skin biopsies were plated in

1-well of a 6-well culture dish with IxMedia. IxMedia consists of 395 mL MEM-alpha

(Gibco/Thermo Fisher Scientific, Grand Island, NY; Cat#: 12571-063;), 50 mL KnockOut Serum

Replacement (Gibco/Thermo Fisher Scientific; Cat#: 10828-028), 50 mL Heat-Inactivated

Human Serum (Innovative Research, Novi, MI; Cat#: IPLA-SERAB-HI), 5 mL GlutaMAX

Supplement (Gibco/Thermo Fisher Scientific; Cat#: 35050-061), 1 mL Primocin (InvivoGen, San

Diego, CA; Cat#: ant-pm-2) and 10ng/mL rhFGF2 cGMP Grade (Waisman Biomanufacturing,

Madison, WI; Cat#: rhFGF). Twenty-four hours prior to transduction, the media were switched to

xeno-free, serum-free, IxMedia. The following day, cells were transduced with non-integrating

Sendai viral vectors 4-7 driving expression of OCT4, SOX2, KLF4 and c-MYC at an MOI of 3

(Thermo Fisher Scientific; CytoTune-iPS Reprogramming Kit) in Viral Transduction Media

(serum free IxMedia plus 10 μM Y-27632 ROCK Inhibitor (EMD Milipore, Billerica,

MA)). Transduction media were removed 18 hours later and replaced daily with serum free

IxMedia. Five days post-transduction, fibroblasts were passaged onto fresh xeno-free rhLaminin

521 (LN521)-coated 10 cm culture dishes (Corning Life Science, Tewksbury, MA) and fed with

fresh serum free IxMedia plus Revita Cell (Thermo Fisher Scientific). On day 6, the media were

transitioned to xeno-free Human Essential 6™ (Thermo Fisher Scientific) using equal parts

Essential 6™ and serum free IxMedia. From day 7 to day 21 cultures were fed daily with fresh

Essential 6™. On day 21, cultures were transitioned to Human Essential 8™ iPSC maintenance

media (Thermo Fisher Scientific) and fed daily. When iPSC colonies reached 1-2 mm in

diameter they were manually isolated and passaged onto fresh 12-well rhLaminin521-coated

culture plates and clonally expanded in Human Essential 8™ media. Following expansion, cells

were analyzed for pluripotency (i.e., expression of endogenous pluripotency factors) via rt-PCR

and loss of transgene expression (i.e. lack of detectable expression of OCT4, SOX2, KLF4 or c-

MYC) using a qPCR-based scorecard assay 8,9.

Patient 2:

500,000-800,000 patient-specific dermal fibroblasts were nucleofected with 1ug of each of the

three episomal plasmids (Addgene plasmids #27077, #27078, #27080), and reprogrammed and

characterized as described previously 10.

Differentiation of patient-specific iPSCs: Puromycin-selected patient-derived iPSCs (see above)

were cultured on ultra low-binding plates (Corning Life Sciences) in embryoid body formation

medium [DMEM F-12 (Thermo Fisher Scientific), 10% knockout serum replacement (Thermo

Fisher Scientific), 2% B27 supplement (Thermo Fisher Scientific), 1% N2 supplement (Thermo

Fisher Scientific), 1% L-glutamine (Thermo Fisher Scientific), 1X NEAA (Thermo Fisher

Scientific), 0.2% Primocin™ (Invivogen), 1 ng/ml Dkk-1 (R&D Systems, Minneapolis, MN), 1

ng/ml IGF-1 (R&D Systems), 1 ng/ml Noggin (R&D Systems) and 0.5 ng/ml bFGF (R&D

Systems)] for 4-5 days. The resulting embryoid bodies (200-300/well) were plated on 6-well

plates (Corning Life Sciences) coated with 25 ug/ml collagen (BD Bioscience, San Jose, CA) 50

ug/ml laminin (Thermo Fisher Scientific), and 100 ug/ml fibronectin (Sigma-Aldrich, St. Louis,

MO) and cultured in differentiation media one [DMEM F-12 (Thermo Fisher Scientific), 2% B27

supplement (Thermo Fisher Scientific), 1% N2 supplement (Thermo Fisher Scientific), 1% L-

Glutamine (Thermo Fisher Scientific), 1X NEAA (Thermo Fisher Scientific), 0.2% Primocin™

(Invivogen), 10 ng/ml Dkk-1 (R&D Systems), 10 ng/ml IGF-1 (R&D Systems), 10 ng/ml Noggin

(R&D Systems) and 5 ng/ml bFGF (R&D Systems)] for ten days. The embryoid bodies were

then differentiated for six additional days in differentiation media two [(differentiation media one

plus 10 uM DAPT (EMD Millipore)] followed by an additional 12 days of culture in differentiation

media three [(differentiation media two plus 2 ng/ml aFGF (R&D Systems)]. The resulting

human photoreceptor precursors were cultured for an additional 60 days in differentiation media

four [DMEM F-12 (Thermo Fisher Scientific), 2% B27 supplement (Thermo Fisher Scientific),

1% N2 supplement (Thermo Fisher Scientific), 1% L-Glutamine (Thermo Fisher Scientific), 1X

NEAA (Thermo Fisher Scientific), 0.2% Primocin™ (Invivogen)].

rAAV vector production:

rAAV5 vectors expressing human U6 promoter-driven sgH23-2 and humanized S. aureus Cas9

under the control of the Cbh promoter were generated by standard triple transfection 11,12 of

HEK293T cells (ATCC) and purified using an iodixanol step gradient (Sigma) and anion

exchange column (GE Healthcare). Titers were determined by qPCR.

Animals and subretinal injections: All procedures were approved by the Institutional Animal Care

and Use Committee of the University of Iowa and Iowa State University and were conducted in

accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision

Research. Handling and preparation of the animals for the procedures were done by

experienced animal technicians. 7 week old non-immune suppressed Pro23His mutant retinal

degenerative mini swine (64-1 founder 13) received AAV2/5-sgH23-2-SaCas9 unilaterally. The

contralateral eye served as a control. Each animal underwent pars plana vitrectomy using a 23-

guage instrument, including induction of posterior vitreous detachment, under general

anesthesia. A 41-guage flexible polyamide cannula was used to create a small retinotomy

allowing 300 ul subretinal blebs containing 2.5 x 109 TU vector each. Sclerotomies were sutured

and each eye was rinsed in 5% povidone-iodine. Three weeks post injection, eyes were

enucleated and a 20mm section encompassing the injection site was dissected using a biopsy

punch and flash frozen prior to genomic DNA isolation using the NucleoSpin Tissue Kit

(Clontech) according to manufacturer’s instructions. The surgeons and researchers performing

the post-harvest data collection were masked to treatment condition.

Supplementary References

1. Cong, L, Ran, FA, Cox, D, Lin, S, Barretto, R, Habib, N, et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819–823.

2. Ran, FA, Cong, L, Yan, WX, Scott, DA, Gootenberg, JS, Kriz, AJ, et al. (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature 520: 186–191.

3. Yin, YC, Boone, M, Meuris, L, Lemmens, I, Van Roy, N, Soete, A, et al. (2014). Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations. Nat. Commun. 5: 4767.

4. Small, KW, DeLuca, AP, Whitmore, SS, Rosenberg, T, Silva-Garcia, R, Udar, N, et al. (2016). North Carolina Macular Dystrophy Is Caused by Dysregulation of the Retinal Transcription Factor PRDM13. Ophthalmology 123:9-18.

5. Tucker, BA, Mullins, RF, Streb, LM, Anfinson, K, Eyestone, ME, Kaalberg, E, et al. (2013). Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa. Elife 2: e00824.

6. Tucker, BA, Anfinson, KR, Mullins, RF, Stone, EM and Young, MJ (2013). Use of a synthetic xeno-free culture substrate for induced pluripotent stem cell induction and retinal differentiation. Stem Cells Transl Med 2: 16–24.

7. Tucker, BA, Park, I-H, Qi, SD, Klassen, HJ, Jiang, C, Yao, J, et al. (2011). Transplantation of adult mouse iPS cell-derived photoreceptor precursors restores retinal structure and function in degenerative mice. PLoS ONE 6: e18992.

8. Fergus, J, Quintanilla, R and Lakshmipathy U. (2016). Characterizing pluripotent stem cells using the TaqMan® hPSC Scorecard™ panel. Methods Mol. Biol. 1307: 25-37.

9. Tsankov, AM, Akopian, V, Pop, R, Chetty, S, Gifford, CA, Daheron, L, et al. (2015). A qPCR ScoreCard quantifies the differentiation potential of human pluripotent stem cells. Nat. Biotechnol. 33: 1182-1192.

10. Schlaeger, TM, Daheron, L, Brickler, TR, Entwisle, S, Chan, K, Cianci, A, et al. (2015). A comparison of non-integrating reprogramming methods. Nat. Biotechnol. 33: 58-63.

11. Brument, N, Morenweiser, R, Blouin, V, Toublanc, E, Raimbaud, I, Cherel, Y, et al. (2002). A versatile and scalable two-step ion-exchange chromatography process for the purification of recombinant adeno-associated virus serotypes-2 and -5. Mol Ther. 6: 678-686.

12. Matsushita, T, Elliger, S, Elliger, C, Podsakoff, G, Villarreal, L, Kurtzman, GJ, et al. (1998). Adeno-associated virus vectors can be efficiently produced without helper virus. Gene Ther. 5: 938-945.

13. Ross, JW, Fernandez Castro, J, Zhao, J, Samuel, M, Walters, E, Rios, C, et al. (2012). Generation of an inbred miniature pig model of retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 53: 501-507.

Supplementary Figure Legends

Figure S1. Screening of sg1MAK. A) Histogram showing percent NHEJ in HEK293T cells

transfected with each guide. Efficiency was quantified by subcloning and Sanger sequencing

(please refer to Methods for further detail). Data are represented as mean ± S.E.M. N = 3. B)

Sequence alignment of selected clones from HEK293T cells transfected with sg1. Arrow

indicates predicted cleavage site. The protospacer adjacent motif (PAM) sequence is

underlined. C) Representative gel image confirming homologous recombination of donor

plasmid containing wild-type MAK exon 9 in HEK293T cells. D and E) Representative gel

images of T7E1 assays in HEK293T (D) or corrected iPSC clone 6 (E) cells for each off-target

locus tested. P indicates target is in protein-coding region.

Figure S2. Off-target analysis (CEP290). (A) Off-target analysis showing sequence

alignments of off target locus PCR amplicons for clone 12 and clone 3 (derived using the single

guide strategy). Expected sequencing results are shown above each chromatogram (potential

off-target sites are underscored). Note that the G1OT4 locus (intronic region of DNAJC6)

appears to be heterozygous in this patient, with one allele containing a pre-existing (before

CRISPR treatment) single nucleotide insertion. (B) Off-target analysis of guide 1 (orange) and

guide 2 (blue) for clone 88 using the dual guide strategy. Unaffected indicates that the predicted

off-target site is unchanged in the respective clone.

Figure S3. Screening of CEP290 guide pairs. A) Representative gel image (left) of the target

locus amplified via PCR in HEK293T cells transfected with plasmids expressing each guide pair

and S. aureus Cas9 (SaCas9). B) Sequence confirmation of genomic correction in iPSC clone

transfected with a plasmid expressing guide pair sg4/5 and our donor construct. Amplicons were

gel purified, subcloned, and sequenced via Sanger sequencing. Exon X sequence is underlined.

The IVS26 nucleotide is in bold.

Figure S4. Off-target activity of IVS26 guides 4 and 5. A) Representative gel images of T7E1

assays in HEK293T cells for each sg4 guide off-target locus tested. OT12, non-protein-coding

target, was omitted due to lack of available cell lines with a Y chromosome. B) Representative

gel images of T7E1 assays in HEK293T cells for each sg5 guide off-target locus tested. Any

digested products observed were the result of homogeneous stretches of sequence within the

amplicon. P indicates target is in protein-coding region. C) Representative gel images of T7E1

assays in corrected patient-derived clones 4-11 and 4-5 for each of sg4 (top) and sg5 (bottom)

guide off-target locus tested.

Figure S5. Off-target activity of sgH23-2. A) Representative gel images of T7E1 assays in

HEK293T cells for each off-target locus tested. Any digested products observed were the result

of homogeneous stretches of sequence within the amplicon. P indicates target is in protein-

coding region. OT21 was added to the analysis because the OT19 locus could not be

determined using each of five different primer sets in two cell lines. B) Diagram of the RHO

locus where the Pro23His mutation (red box) occurs and the location of sgRNAs tested. C)

Histogram showing percent NHEJ in HEK293T cells transfected with each guide. Data are

represented as mean ± S.E.M. N = 3. Efficiency was quantified by subcloning and Sanger

sequencing (please refer to Methods for further detail). D) Representative gel images of T7E1

assays in corrected patient-derived clones 3-3 and 5-2 for sg3 guide off-target locus tested.

Supplementary Tables

Table S1. Primers employed in these studies.

Name Sequence (5'-3') Figure On target primers MAK1-1F AGGAGGACACTAAGGCTTTGAGAAG 1 MAK1-2R ACGCCTCCTACCACTTTTATGACTC 1 CEP290gt-F GTGTGCACCACCACACCAGCT 2 CEP290gt-R GGCAGTAAGGAGGATGTAAGACTGG 2 CEP290gt-SEQ CACCTTGTTAGCCAGGATGGT 2 hsIVS26F ATGTCCTCTGTCCTATCCTCTC 3 hsIVS26R CTCAGCTAGTTCATCTCTTGCTC 3 P23HsurvF GGACAGACAAGTCATGCAGAAG 4 P23HsurvR ATCCATGCAGAGAGGTGTAGAG 4 MAKHDRF AACTTGTTTATTGCAGCTTATAATG 1 MAKHDRR TCTAAGTTTCCAAAAGCCCTCACC 1 EXONXF CCATCATGCCCGGCTAATTT 3 EXONXR CTAAGACACTGCCAATAGGGATAG 3 IVS26TaqF CTCCTAAAGTGCTGGGATTACAGAT 3 IVS26TaqR ACTGCCAATAGGGATAGGTATGAGAT 3 IVS26TaqProbe FAM-CAGGTGCGGTGGCTCA-NFQ 3 rt-PCR primers MAKrtF CCCTTCGTCAAATCATCTGG 1 MAKrtR TCTTGTTTTCCCCTGTCGAG 1 CEP290E26-F GGCCATTTTCAAGATTGCAGCT 2 CEP290E27-R AGTTTCCTGTTCCCAGGCTTG 2 CEP-CryX-R ACCATCCTGGCTAACAAGGTG 2 CEP290rtF GTTGTCAATGGAGGCTGAAGT 3 CEP290rtR TGTTCCCAGGCTTGTTCAATAG 3 POLR2AF TAACCTGCTGCGGATGATCTGGAA 3 POLR2AR GATGTTGAAGAGCAGCGTGGCATT 3 Off-target primers sg1MAK_OT1_F CCCTGATGCTGACGATTACTT S1 sg1MAK_OT1_R GGTGCCCTATCCAAAGGTATT S1 sg1MAK_OT2_F TCCAGAGAAGCAGAGACTTAGA S1 sg1MAK_OT2_R GGTATCCAGGCCAAACCATTA S1 sg1MAK_OT3_F CAGAGGCTGAAGTGAAGTTACA S1 sg1MAK_OT3_R ACCATGTAAGTGGTCCAACTAAG S1 sg1MAK_OT4_F CTCGTCCTTGAATAGGACAACTC S1 sg1MAK_OT4_R ATGATCACACCACTGCACTC S1 sg1MAK_OT5_F CAGGCTCTACTGTGCAGTTAT S1

sg1MAK_OT5_R CCTTGGGAAGGCTTCACTTA S1 sg1MAK_OT6_F GAGGTTGGAGGATTGCTTGA S1 sg1MAK_OT6_R CACATTCCATTAGGATGGCTAGA S1 sg1MAK_OT7_F CCAGGCACTCAGTAGAAGTTAG S1 sg1MAK_OT7_R GGGAGAGAGAGAGAGAGAGAAA S1 sg1MAK_OT8_F TGGTGAAAGGAAGGGTGATTAG S1 sg1MAK_OT8_R CCCACATGTCAAGGGAGAAA S1 sg1MAK_OT9_F GTCTGGCTTTGTGTTTGTTCTT S1 sg1MAK_OT9_R GCTCCTTCCTCTTTCTCCTTATC S1 sg1MAK_OT10_F GACATTCTTCCACCTCAGTCTC S1 sg1MAK_OT10_R GTGCAAATACCCTGTTCCTTTATC S1 sg1MAK_OT11_F ACAGGGATTGTGCCTTAGTG S1 sg1MAK_OT11_R ACTAGCAGTGAGAGGGTAGTT S1 sg1MAK_OT12_F CCTGTCCAGAGTCACAAGAATC S1 sg1MAK_OT12_R TGGGTGGTTTGGCCTTTATC S1 sg1MAK_OT13_F CCCAGACAAAGGTGGAAGAA S1 sg1MAK_OT13_R AGAAGGAGCAGAAGAAGGAATG S1 sg1MAK_OT14_F GGTGGTGTCTGCTCAAAGTA S1 sg1MAK_OT14_R CCCATGCCCAAGGTTACATA S1 sg1MAK_OT15_F GAGGCAGCAGAATCACTTCA S1 sg1MAK_OT15_R CCCTGACTCTGTGACAACATAC S1 sg1MAK_OT16_F CTCCCTGGTTGTCCTTTCTATA S1 sg1MAK_OT16_R GAGGTGGCTCATACCTGTAATC S1 sg1MAK_OT17_F AGGCTTGTCTTCAGAGTCTTTC S1 sg1MAK_OT17_R GGACCTAGAATCAAGGGTCAATC S1 sg1MAK_OT18_F TCAACCTTGCCATCAACAAATC S1 sg1MAK_OT18_R ATGGGATGTCCACTTGGTTC S1 sg1MAK_OT19_F GTGAGGACTGTGGGTTTCTT S1 sg1MAK_OT19_R GTGAGTGAGCAAGAGGGTAAT S1 sg1MAK_OT20_F CTCCACTGCAAGCGTAAGA S1 sg1MAK_OT20_R GGAAAGCCAGTCAGCCTATAA S1 G1OT1-F GGATCCCAGTCCTACTCTGGT S2 G1OT1-R TGACTGTGTGAATGTTTCAAGC S2 G1OT1-Seq GAACATGGCCCCAGCAACCAAA S2 G1OT2-F CCCGGGTTCAAGCCATCCTCCC S2 G1OT2-R GGGTGAAACACCCCCATGATTC S2 G1OT2-Seq GTGGAGATGGGGTTTCTCTGTG S2 G1OT3-F GTGTACTAGGCTCTAGGCCAAAC S2 G1OT3-R GTGATCTGTATCCTTGTTATCC S2 G1OT3-Seq GAAAGGAAATTGAGCCTTGAAAA S2 G1OT4-F GGAGACGAAGGGTGTGTGACATG S2

G1OT4-R GTTGGCGAGAGAACCCAGGACG S2 G1OT4-Seq AGCAGGAGAAAGGCTTATATAGGTC S2 G1OT5-F AAGCCCATCAGGGACAAAGG S2 G1OT5-R GGGAGAGGGCGCTCTAGTAT S2 G1OT5-Seq CAGATTTGGTGGTTTGCTCCCC S2 G1OT6-F GAGGTGGTAAAGTTGCTATCCAG S2 G1OT6-R CATCTGTTGGCCATTTGTATGTC S2 G1OT6-Seq GGTTGCCTGAGAGCAATAGGTAG S2 G1OT7-F CCGTCATCTACGTGGTTCCCTCG S2 G1OT7-R CTTCACGGAAGTATTGAACGGGTG S2 G1OT7-Seq ATGCTAGCTAGCTGCTGTTACAA S2 G2OT1-F AACTGCTAGCCTCAAGCGAT S2 G2OT1-R GACTCCCACGCCTAAATCCC S2 G2OT1-Seq TGCTCCCTTTCTGAGCAAAGAT S2 G2OT2-F AACTTCTCTGAACCTCTGTCCT S2 G2OT2-R GGCATGAAGTTGTAGCTGTGT S2 G2OT2-Seq ACATAATATGCCTGGTGCAG S2 G2OT3-F TCCAGGACCACAGAATCAAGAC S2 G2OT3-R TGCCTATGGGGTGACCAGAC S2 G2OT3-Seq GGACCTCAACATTAGATGCAG S2 G2OT4-F CATGGTTCGGTAAGTACTGGG S2 G2OT4-R AGCTGTGCTTTGTTGCAGTCT S2 G2OT4-Seq CATGGTTCGGTAAGTACTGGG S2 G2OT5-F TCCTTCTGCAGACTCCATTGAA S2 G2OT5-R CTTAACACTGGGCAGTTCCG S2 G2OT5-Seq CCATGCAAGATGTATGTTGT S2 G2OT6-F AAAGTTCAATTCTTCCCTGC S2 G2OT6-R CTCCTGAAGCCACACAGCTAA S2 G2OT6-Seq GCAGATTTTAGACTTGCTGGC S2 G2OT7-F AGCACTCGGCATAGTACCCA S2 G2OT7-R ATGTCCACCTTCCAAAGCCA S2 G2OT7-Seq TACTTGCAGAGCCATTTTGGG S2 G2OT8-F GCATGTTAATCTACCTTTTGCTTTC S2 G2OT8-R CCCGGCCAGCAGATATTTTTTAAAG S2 G2OT8-Seq GCCATTATTTGTTTTACCAGTGC S2 G2OT9-F CCCCGACTAGTTTGTCCCAC S2 G2OT9-R GGGCCTGCCGTGGTATATTT S2 G2OT5-Seq CTGCAACAACTGTGGCTTCT S2 sg4OT1_F CACGGTTGTTGCGAAGATTG S3 sg4OT1_R GTTGCAGGCTACAGAAGTCTAA S3 sg4OT2_F GTGGTGGTCCCATAAGGTTAAT S3

sg4OT2_R TTTAGTCTGGCATGGTCAAGAG S3 sg4OT3_F GATGAAGACAGGGCAACAAATC S3 sg4OT3_R GCCTGGTCTCCTGGAAATAAA S3 sg4OT4_F GGTTGGCCTCAATAGGGTTAAG S3 sg4OT4_R TCCGAAGTAGCTGGGAATACA S3 sg4OT5_F GACCACCATCTCTTGCCATAA S3 sg4OT5_R GGTCCCTCCACAGGAAATAAA S3 sg4OT6_F CACAGAAACTCTCGGGCTAAA S3 sg4OT6_R CCTCCCAAAGTGCTAGGATTAC S3 sg4OT7_F CCCTTGTCTTGTTGCTTCTCT S3 sg4OT7_R CCTCTCAAAGTGCTGGGATTAC S3 sg4OT8_F TTCCCTGGGAGTCTCTTCTT S3 sg4OT8_R CCTACTGTGTGCCAGGTATTG S3 sg4OT9_F CAGAGCAAGACTCCATCTCAAA S3 sg4OT9_R AATCCCAGGTCTGTCACTTAAC S3 sg4OT10_F GATGTCACACTGGTAGCCTAAA S3 sg4OT10_R GTCCCACATCTTTCTCTTCTCTC S3 sg4OT11_F CCCAGCAATTTCACTCCTACAT S3 sg4OT11_R CCGCCTAGCTCTTGGTAAAC S3 sg4OT13_F GCAGATGTGTGAGGAGGAATAA S3 sg4OT13_R GTATCACCATGCCCACCTAAT S3 sg4OT14_F GAGAAACCCAGTCTCTGCTAAAA S3 sg4OT14_R ACAGAACAGACAGAGGGACTAA S3 sg4OT15_F GGGAGGGATCAAAGAACATGAG S3 sg4OT15_R ACAACATGGCTAGGTGTAGTG S3 sg4OT16_F GTACCTGCCTCAGTAGCTTATTT S3 sg4OT16_R CCAGGCCAGTCACATCTTATAG S3 sg4OT17_F GGCGGGTGGTATTGTTTCTA S3 sg4OT17_R ACTCTGGCTTTAGGGTGAATG S3 sg4OT18_F GACTTCCTTCCCTCCAGTTTACC S3 sg4OT18_R TGTTTAGCACCCAGTTGATCTAT S3 sg4OT19_F TGAGAGGGTCCTCATACCTTACC S3 sg4OT19_R GCCTGTAATCCCGACACTTT S3 sg4OT20_F TCAACCCTTGAGCCAGTTATTC S3 sg4OT20_R ACAGTCTATGTCTTGGCGTATTG S3 sg4OT30_F CCTTCACCCAGCTACACTTTAC S3 sg4OT30_R GAGATCCACCTGCTTCAGATTT S3 sg5OT1_F CTTTCCAGCCCTGACCTATTC S3 sg5OT1_R AAGCATCTATTGGGTGTCTACTT S3 sg5OT2_F GAGTATTCTCCATGTGCCTTACA S3 sg5OT2_R TTCTCACTGCCTCCTCATAAATC S3

sg5OT3_F GCACGACCAGAGGGAATAAA S3 sg5OT3_R CATCCCAGCTTACATCTATCGAG S3 sg5OT4_F GACAGCTAGTGGCAGATTAAGG S3 sg5OT4_R TGCCTAGCCCAAAGGTTTAC S3 sg5OT5_F CTTTGGCACCCATTTCAACATA S3 sg5OT5_R CAGTCTTCACAAACAGCAAGTC S3 sg5OT6_F ACCACAAACACATGGGACATA S3 sg5OT6_R GCCACTTCAGCATCCTTTAGA S3 sg5OT7_F GTACCACAGAAAGAGGACAGAATAG S3 sg5OT7_R TCCAGGTGCTTTGGAAGAATAG S3 sg5OT8_F GGTAGGAAGAAGAGTGTGTGTG S3 sg5OT8_R CAATTGCAGATGATGCCAAGAG S3 sg5OT9_F CGCTGGCTTCAGACAGATAA S3 sg5OT9_R CGCCTCAGTTCACGAACTATAA S3 sg5OT10_F ACAGGGTTTCACTCTGTCATC S3 sg5OT10_R CACCTGTAATCCCAGCACTT S3 sg5OT11_F CTGCAACTACTCCCACTCTATC S3 sg5OT11_R CCCTGTTCCTTCACTCTCATT S3 sg5OT12_F TTGTTGGACACCTGAGCTATTT S3 sg5OT12_R AGGTCCCAAGTTATCTGCTTTG S3 sg5OT13_F CTTCCTCCCTCCTGACAGATA S3 sg5OT13_R ACGAGGACACAGGAGAATAAAC S3 sg5OT14_F ATGGGAGCAGTGTGGTATAATG S3 sg5OT14_R GTCCAATGCCAGTGTACTGAT S3 sg5OT15_F CAGGAAGCCCAGTCAAAGAT S3 sg5OT15_R CAAAGTTCAAAGGTGTGAGGTAAG S3 sg5OT16_F GCCTGTAATCCCAGCACTTT S3 sg5OT16_R TACCGAGTTGCAGGTCTTCT S3 sg5OT17_F GGAACACATCCCAGGGTATTTA S3 sg5OT17_R GTAGTCAATCCATCCTCCAAACT S3 sg5OT18_F CTCCTGCCTATGCTCTGTATTG S3 sg5OT18_R CTGTGCCAGGTAGTGGATTT S3 sg5OT19_F GAAATGGCCCTGGACTTAACTA S3 sg5OT19_R AACAAACCACATGCAGACTAAAC S3 sg5OT20_F ACAAGCCCATAGAGGGATAGA S3 sg5OT20_R GACAGAGCGAGACTGTCTTAAA S3 sg5OT33_F CCACACAAGGAGAACCCAAT S3 sg5OT33_R GGAAATCCAAATCCACCAGTTTC S3 sgH23-2OT1_F CTCAGTGTTAACCACAGCTACA S4 sgH23-2OT1_R GGGCTGAGAGAGAAAGTAATGAG S4 sgH23-2OT2_F GGAAGCAGGACCAGTACAAA S4

sgH23-2OT2_R AGAGCAGTGGAGAAGGTAGA S4 sgH23-2OT3_F AGGAGTGAAAGGGAGCAATG S4 sgH23-2OT3_R CCAGCAGCTGAGATCCTTAAA S4 sgH23-2OT4_F GTGCAGGTGGGAGGTATTG S4 sgH23-2OT4_R CTGCTGTGCCGTAGAAACT S4 sgH23-2OT5_F CACCACCACACTTGGCTAAT S4 sgH23-2OT5_R ATTCTGCCAGCCAGGAGTAGTATAA S4 sgH23-2OT6_F GGTTGACTGTACCCTCTGATTT S4 sgH23-2OT6_R GATCTGGAAGCTGATACGTGAG S4 sgH23-2OT7_F CACCTTCCCAGATTCCTTCAA S4 sgH23-2OT7_R CACTCTTGTGCCTCCTTCTAC S4 sgH23-2OT8_F CCATCCTAGTGAGGGTGAAATG S4 sgH23-2OT8_R AATGGTGGTCCTCAAAGGTAAG S4 sgH23-2OT9_F GAAGCACAGAGAGGGAGATAAAC S4 sgH23-2OT9_R CCCTAAGTTCCCGTCTGTATTG S4 sgH23-2OT10_F TGCACTTCTTACACCCATGAA S4 sgH23-2OT10_R CCCACATGTCAGCTCTGTTTA S4 sgH23-2OT11_F GTATGGAAGGATGGACAAGTGAG S4 sgH23-2OT11_R CCTGATGGACAAGGCAATTAGA S4 sgH23-2OT12_F AGGCCTCAAGTGATCTGAATG S4 sgH23-2OT12_R CTCTGTAACTGAGGTCCAACAC S4 sgH23-2OT13_F TGAGGAGAACAGCGGAGAA S4 sgH23-2OT13_R TCTGAAGCGCCAGTTTGTAG S4 sgH23-2OT14_F CTGTGGTGGGCTAACAGATAAA S4 sgH23-2OT14_R GGTGATGGTGGTTACACAAGA S4 sgH23-2OT15_F GTATCTCCGCATACCCAACATA S4 sgH23-2OT15_R GCGCCTTTCTCTCCACA S4 sgH23-2OT16_F CACTGTAGATGCCCTGAAGAC S4 sgH23-2OT16_R AGGCAGAATGTCACCAGATAAA S4 sgH23-2OT17_F CCTGCCCTTCCCATTCAATA S4 sgH23-2OT17_R GGGAAAGCGAGAGAGAGAGA S4 sgH23-2OT18_F ATTACCTCAACGTCACCTACAC S4 sgH23-2OT18_R CAGGCTAACTCTAGGGCATTT S4 sgH23-2OT20_F ATTACCTCAACGTCACCTACAC S4 sgH23-2OT20_R CAGGCTAACTCTAGGGCATTT S4 sgH23-2OT21_F GCCTGAGAGCCACTGATTT S4 sgH23-2OT21_R CCTGGGATGGATGGATAGAATG S4 sgH23-2OT24_F AAACAAAGGCCCAGAGAGAG S4 sgH23-2OT24_R GGGTCAGCATCAGGGATTAT S4 sgH23-2OT30_F ACCTCATTGAAGAAGTGGTACAG S4 sgH23-2OT30_R CCCAGGATGTGGTTCTCAATAA S4

Supplementary Table S2. Tabulated list, overall scores, and genomic locations of top off-target genetic loci as predicted by the Optimized CRISPR Design Tool, for all sgRNAs employed in Patient 2 samples. Mismatches are indicated in red and PAM matches in bold.

Name sgRNA SEQUENCE Score Locus 1 G1OT1 sgRNA 1 TTTCTCGTAACTATCCCTATAGG 1.7 chr15: -37996642 2 G1OT2 sgRNA 1 TGTCACATTCCTATCCCTATCAG 1.6 chr14: -82047603 3 G1OT2 sgRNA 1 GCTTTCATATCTATCCCTATGGG 1.3 chr5: +123508838 4 G1OT4 sgRNA 1 GAGCACATGCCTATCCCTATTGG 0.8 chr1: -65834054 5 G1OT5 sgRNA 1 AACCCCATTCCTATCCCTATGAG 0.8 chr14: -73279627 6 G1OT6 sgRNA 1 GATTTCATAAATATCCCTATGGG 0.7 chr17: -53208742 7 G1OT7 sgRNA 1 CAGCTCATAACTATCCCTAGTGG 0.6 chr8: -103602219 8 G2OT1 sgRNA 2 GAAATAGTCTCAATTACAACTAG 1.6 chrX: +32100823 9 G2OT2 sgRNA 2 GAAAGATCCACAATTACAACCAG 0.9 chr2: +165206842 10 G2OT3 sgRNA 2 AAAAGTCTCACAATTACAACTAG 0.8 chr10: -88627329 11 G2OT4 sgRNA 2 GAGCTCCTCACAATTACATCTGG 0.6 chr5: +96419596 12 G2OT5 sgRNA 2 GAACTACACACAATTATAACCAG 0.6 chr2: +36088495 13 G2OT6 sgRNA 2 TAGATACACGCATTTACAACAGG 0.5 chr4: +56156328 14 G2OT7 sgRNA 2 AAGAAACTGACAATTACAAAAGG 0.4 chr8: -98403547 15 G2OT8 sgRNA 2 CATATACTTACAATTACAATGGG 0.4 chr4: -165515233 16 G2OT9 sgRNA 2 GAAATGCACACAATTACAAACGG 0.4 chr15: -32465287

Supplementary Table S3. Tabulated list, overall scores, and genomic locations of top off-target loci as predicted by the Optimized CRISPR Design Tool for sg1MAK employed in Patient 1 samples or those predicted by the Benchling platform for sg4CEP290, sg5CEP290 (Patients 3 and 4), and sgH23-2 (Patient 5). Bolded entries indicate sites within protein-coding regions. CDS – coding sequence, ON – on target.

target sequence PAM score locus

CDS

sg1MAK GATAGTAGCTTGTGCATGAA AGG 78.0 chr6:+10792160 ON

OT1 GCTTTTATCTTGTGCATGAA GAG 1.4 chr15:+100211884 N

OT2 TAGAATAGGTTGTGCATGAA GAG 0.8 chr6:+80380408 N OT3 AATAATAGTGTGTGCATGAA GAG 0.8 chr16:-17875088 N OT4 GGGAGTAGTGTGTGCATGAA AAG 0.8 chr4:+34238155 N OT5 GAAAAGAGCCTGTGCATGAA TAG 0.8 chr2:-64413496 N OT6 CATAGCAGCTTGTGCATGCA TAG 0.7 chr5:-159271864 N OT7 AATGGTAGCGTTTGCATGAA GAG 0.7 chr4:-130012374 N OT8 TATAGTAGCTTTTGCAGGAA CAG 0.6 chr13:+84986434 N OT9 GATGGTATCATGTGCAAGAA TGG 0.5 chrX:+36709382 N OT10 TTTATTAGCTTGTGCATGTA CAG 0.5 chr16:-65995026 N OT11 GATAGTGATGTGTGCATGAA AGG 0.5 chr12:-54733086 N OT12 GAGCTTAGCTTGTGCATGCA AGG 0.5 chr14:-104023153 Y

OT13 CACAGTTGCTTATGCATGAA TGG 0.5 chr13:+102767494 N

OT14 GAGGGTTGCTTCTGCATGAA TGG 0.5 chr5:-16608914 N OT15 GAAAGTTTCTTGTGCATGAG AGG 0.5 chr8:+69605727 N OT16 GATAGAAATATGTGCATGAA AGG 0.5 chr1:+26150990 N OT17 GCTTGGAGCTTTTGCATGAA AGG 0.4 chr3:-170912971 N OT18 GTTAGTAGAATATGCATGAA CAG 0.4 chr5:+174923550 N OT19 GATATAAACTTGTGCATGAT GGG 0.4 chr4:-171180203 N OT20 GATTTTATCTTGTGGATGAA GGG 0.4 chr7:-38970487 N OT21 GTGAGTAGCTCTTGCATGAA TGG 0.4 chr5:-102155988 N OT22 GATAAAAGCCTATGCATGAA TAG 0.4 chr5:+144626158 N OT23 ACTAGAAGCTTGTGCAGGAA TGG 0.4 chr7:+17237180 N OT24 GGTGGCAGCTTGTGCAGGAA GGG 0.4 chr5:-176760042 N

OT25 GAAAGTACCGTGTGAATGAA GAG 0.4 chr12:+105499218 N

OT26 CATATTTGCTTGTGCATGCA CAG 0.4 chr12:-101409055 N OT27 TCTAGTAGTTTGAGCATGAA AGG 0.4 chr17:-26655970 N OT28 CATAGAAGCCTGTGCAGGAA AAG 0.3 chr10:+97624252 N OT29 GAAAGTATTTTGAGCATGAA AAG 0.3 chr12:-89365586 N OT30 GAAAATAGTTTGGGCATGAA AAG 0.3 chr2:-183619918 N OT31 GGTAGTAGTATGTGCATGCA CAG 0.3 chr9:+80191913 N OT32 GATGTTAGCTTATGCAGGAA AGG 0.3 chr3:+163365115 N

OT33 GGTAGAAGCCTGTGCATGGA TGG 0.3 chr13:-80624227 N OT34 GAAAGTAACATGTGCATTAA AAG 0.3 chr11:-103717981 N OT35 GAAAGTGTCTTGTGGATGAA GAG 0.3 chr5:+527614 N OT36 GATAGGTGCATTTGCATGAA GAG 0.3 chr18:-69729293 N OT37 GAGAGGGGCTTGTGCAGGAA TGG 0.2 chr7:-46909077 N OT38 AATAGTAGAATGTGGATGAA TAG 0.2 chr8:-64242694 N OT39 GAAAGCAGCTGTTGCATGAA AAG 0.2 chr7:-154969894 N OT40 GTTAGTGGCTAGTGCAGGAA AAG 0.2 chrX:+120560530 N OT41 GATAACAGCTGCTGCATGAA TGG 0.2 chr10:+50679250 N OT42 GATAAAAGCTATTGCATGAA TGG 0.2 chr1:+215879842 N OT43 GCTAGCAGCCTGTGGATGAA GGG 0.2 chr9:-27664149 N OT44 CATGGTAGCTTGTGCATGCC TAG 0.2 chr7:+36643087 N OT45 AATAGTATATTGTGCATAAA AAG 0.2 chr1:+106505000 N OT46 GATGTTAGCTTTTGTATGAA GGG 0.2 chr5:+167293742 N OT47 AATATAAGCTTGTGCATAAA AAG 0.2 chr13:+81436283 N OT48 GTTAGTAACTTGTGGATGAC TGG 0.2 chr4:-15915498 N OT49 GATGGGAGCTGGGGCATGAA GAG 0.2 chr2:-8797949 N sg4CEP290 GTTTCATTCTGTCACCCAGG CTGGA 83.0 chr12:-88102837 ON OT1 GTTTCATTCTGTCACCCAGG CTGGA 100.0 chr1:-28285394 N OT2 GTTTCACTCTGTCACCCAGG CTGGA 13.7 chr15:+41636182 N OT3 GTCTCACTCTGTCACCCAGG CTGGA 4.0 chr16:71868785 N OT4 GTCTCACTCTGTCACCCAGG CTGGA 4.0 chr16:-58534032 N OT5 GTCTCACTCTGTCACCCAGG TTGGA 4.0 chr2:+232453535 N OT6 GTCTCACTCTGTCACCCAGG CTGGA 4.0 chr19:-52657503 N OT7 GTCTCCTTCTGTCACCCAGG CAGGA 3.4 chr2:-47759174 N OT8 ATCTCATCCTGTCACCCAGG CTGGA 2.7 chr16:+77583669 N OT9 CCTTTATTCTGTCACCCAGG CTGGA 2.5 chr1:-154208438 N OT10 GAGCCATTCTGTCACCCAGG CAGGA 2.3 chr13:-51500357 N OT11 GTTTCACTCTATCACCCAGG CTGGA 2.3 chr20:1999972 N OT12 ATCTCACTCTGTCACCCAGG TTGGA 1.8 chrY:+10647847 N OT13 CTTCCATTTTGTCACCCAGG TTGAA 1.8 chr1:-118920867 N OT14 GTCTCACTCAGTCACCCAGG CTGAA 1.7 chr16:+32625538 N OT15 CTCTCATTCTATCACCCAGG CTGGA 1.7 chr9:+118267305 N OT16 CCTTCTTTCTGTCACCCAGG TAGGG 1.6 chr12:+53813222 N OT17 AGCTCATCCTGTCACCCAGG TGGAA 1.5 chr9:-83629000 N OT18 GATTCACTCTGTCACCCAGA GTGAA 1.3 chr9:+85045421 N OT19 GTCTCACTCTGTCACCCAGA GTGAA 1.2 chr1:+176250840 N OT20 GTGTCATCCTGTGACCCAGG AGGGG 1.2 chr9:-89006151 Y OT21 GTCTCCCTCTGTCACCCAGG CTGGA 1.0 chr8:+102539998 N OT22 GTCTCTCTCTGTCACCCAGG CTGGA 1.0 chr7:-103319113 N OT23 GTCTCTCTCTGTCACCCAGG CTGGA 1.0 chr20:+10331911 N

3 OT24 TATTCACTCCGTCACCCAGG CTGAA 1.0 chr6:-118251230 N OT25 ATCTCATTCTGTCATCCAGG CTGAA 1.0 chr1:+174221928 N OT26 GTTTCACTCTGTCACTCAGG CCGAG 0.9 chrX:+125262092 N OT27 ATTCCATCCTGTCACCCAGC CAGGA 0.9 chr20:+59148799 N OT28 GGGTCTTGCTGTCACCCAGG CTGGA 0.9 chr9:-93931031 N OT29 GTTTCACTCCATCACCCAGG CTGGA 0.9 chr20:-48918037 N OT30 ACCTCATTCTGTCACCCAGA ATGGA 0.9 chr6:-89634395 Y OT31 GTCTCACTCTGTCACCCAAG TTGGA 0.9 chr19:+37690688 N OT32 ATCTGGTTCTGTCACCCAGG AAGAA 0.8 chr15:-55863368 N OT33 CTTTAATGCTGTCACCTAGG TAGAA 0.7 chr1:+163605343 N OT34 ATTTCACTCTGTCATCCAGG CTGAG 0.7 chr10:-22175112 N

OT35 TTCTCATTCTGTCACTCAGG CTGGA 0.7 chr11:+109934178 N

OT36 ATCTCACTCTGTCACCCAGC CTGGA 0.6 chrY:+11003139 N OT37 ATCTCACTCTGTCACCCAGC CTGGA 0.6 chrY:+10894810 N OT38 TTCTCACTCTGTCACCCAGA CAGGA 0.6 chrY:+10809500 N OT39 GGTCCATTCAGTAACCCAGG CAGAG 0.6 chr18:-36285007 N OT40 GTCCTATTCTGTGACCCAGG AAGAG 0.6 chr7:-154987261 N OT41 GTCTCACTCTGTCATCCAGG CTGGG 0.6 chr19:-15507234 N OT42 TTCTCTCTCTGTCACCCAGG GTGGA 0.6 chr13:+96056748 N OT43 GACTCACTCTATCACCCAGG CTGGA 0.6 chr4:+35406090 N OT44 AATTCAATCTGGCACCCAGG CTGAA 0.6 chr7:+8532235 N OT45 GTCTTGCTCTGTCACCCAGG CTGGA 0.6 chr16:-14276959 N OT46 GTCTTGCTCTGTCACCCAGG CTGGA 0.6 chr2:+84999089 N OT47 GTCTTGCTCTGTCACCCAGG CTGGA 0.6 chr5:+172390428 N OT48 GTCTGGCTCTGTCACCCAGG CTGGA 0.6 chr5:+133056395 N OT49 GTCTTGCTCTGTCACCCAGG CTGGA 0.6 chr19:+20929141 N sg5CEP290 GTAAGACTGGAGATAGAGAC AGGAA 54.8 chr12:+88101105 ON OT1 GTAAAACTGGAGATAGAGAG ATGAG 5.7 chr4:-174067925 N OT2 GGCAAACTGGAGATAGAGAC TGGGG 2.4 chr22:+49251828 N

OT3 CCAGGACTGCAGATAGAGAC CTGGA 1.4 chr15:+101064157 N

OT4 TTAGAACTGCAGATAGAGAC TTGAA 1.4 chr22:-33227672 N OT5 GATATACTGTAGATAGAGAC CAGAA 1.4 chrX:-99740624 N OT6 GGAAGACTGAAGATAGAGCC TTGAA 1.2 chr6:-122112228 N OT7 TAAATACTAGAGATAGAGAC ATGGA 0.9 chr3:-60561082 N OT8 GAAAAACCTGAGATAGAGAC CTGAG 0.9 ch7:-114946383 N OT9 CTATGACTTAAGATAGAGAC TAGAA 0.9 chr4:-13965911 N OT10 AAAAAACTGGAGATAGAGAA AGGAA 0.9 chr17:+55526015 N OT11 GCCAGGCAGGAGATAGAGAC AAGGA 0.9 chr5:+158176160 N

OT12 GAATGGCTGCAGATAGAGAC AAGAG 0.9 chr15:-69485997 N OT13 TTCATGCTGGAGATAGAGAC CTGAA 0.8 chr9:+115445631 N OT14 GTAGCACAGGAGATAGAGAT GAGAA 0.8 chr1:+97103176 N OT15 GTAAGCCAGGAGATAGGGAC AGGAA 0.7 chr17:+27200273 N OT16 GCAAGACTGAAGATAGAAAC AGGAG 0.7 chr6:-16451897 N OT17 GTAGAACTGAAAATAGAGAC TGGGA 0.7 chr6:+72176542 N OT18 GAAGGACAGGAGGTAGAGAC AGGAA 0.7 chr13:+46879555 N OT19 ACAAGACTGAAGGTAGAGAC AAGAA 0.6 chrX:-72001711 N OT20 GGGAGGGTGGAGATAGAGAC GGGAG 0.6 chr19:-3040199 N OT21 CTAAGAATGAAGATAGAGAT GCGAA 0.6 chr8:+53592506 N OT22 GTAAGACTGGAGGTTGAGAC GAGAA 0.6 chr10:+15608931 N OT23 ATAAGAGTGTATATAGAGAC ATGAA 0.5 chr7:+87888982 N OT24 GTGAGAAAGGAAATAGAGAC AGGGA 0.5 chr2:-8540173 N OT25 GTCAGACCAGGGATAGAGAC GGGGA 0.5 chr2:-225731275 N OT26 AGAAGACTGGATATAGAGAG AAGAG 0.5 chr13:+37477297 N OT27 GGAAGACAGGTGATAGAGAG CTGAG 0.5 chr13:-37477297 N OT28 GAAGGACGGGAGATGGAGAC TAGGG 0.5 chr1:+43276004 N OT29 GAAAGAAAGGAGATAGAGGC AGGAG 0.4 chr6:-132793409 N OT30 CTGAGACTGGAGATAGGGAA GGGGA 0.4 chr10:-73868169 N OT31 ATGAGCCTGGAGATAGAGGC AAGGG 0.4 chr5:-146599549 N OT32 GCAAGACTGAAAATAGAGAG CTGGG 0.4 chr8:+85338060 N

OT33 GCAGGGCTGGAGGTAGAGAC CTGAA 0.4 chr4:-99623521 Y

OT34 ATATGACTGGTGATAGAGTC CTGAA 0.4 chr4:-185813101 N OT35 GGATGTCTGGAGATAGAGGC CTGGG 0.4 chr11:-62614215 N OT36 GAACAACTGGAGATAGACAC ATGAG 0.4 chr6:-162875922 N OT37 GCAGGACTGGAGGTAGAGAG AAGAA 0.4 chr10:-124956403 N OT38 GAAATACTGGAGTTAGAGAT GTGGA 0.4 chr6:+169977753 N OT39 GTACAGCTGGAGATAGAGGC GTGGG 0.4 chr14:+99754594 N OT40 TTAAGAATAGAAATAGAGAC ACGAA 0.3 chr3:-27910115 N OT41 CTAAGACTGCAGATAGGGAG AGGAA 0.3 chr8:+51613066 N OT42 CTAAGCATGGAAATAGAGAC TTGGA 0.3 chrX:+63852426 N OT43 GTAAGCTTTCAGATAGAGAC TTGAG 0.3 chr4:+87067670 N OT44 GGAAGACTGGAGATACAGAG AGGAA 0.3 chr5:-15707893 N OT45 GAAAGGCTAGAGATAGAGAA GGGGA 0.3 chr22:+32765706 N OT46 GTAACCTTGGAGATAGAGAA TTGAG 0.3 chr2:+48320008 N OT47 CTAAGAGTGCAGATGGAGAC ATGAG 0.3 chr20:-5502072 N OT48 CTAAGAGTGCAGATGGAGAC ATGAG 0.3 chr20:+5473426 N OT49 GGAAGGCTGGAGAAAGAGAC TTGAA 0.3 chr9:+116415697 N sgH23-2 GGGTGTGGTACGCAGCCACT TCGAG 100.0 chr3:+129528801 ON OT1 GTGTGTGGGATGCAGCCACT TGGGA 1.0 chr8:+61659010 N OT2 AGGGGTGGTCAGCAGCCACT CTGAA 0.9 chrX:-40167010 N

OT3 GGTGGTGATATGCAGCCACT CAGGA 0.9 chr5:+142721194 N OT4 AGCTGTGGTCAGCAGCCACT CAGGG 0.9 chr22:+46488193 N OT5 GCGTGTATTATGCAGCCACT TTGGG 0.6 chr11:+64945888 N OT6 GGGTGTAGAATGCAGCCACT TAGAA 0.6 chrX:-36777359 N OT7 TTGTGTGGGAGGCAGCCACT GGGAG 0.6 chr6:+23981176 N OT8 GGATGTGTAATGCAGCCACT GAGAG 0.5 chr12:-128197383 N OT9 GGTTGTGAAATGCAGCCACT CTGGA 0.5 chr12:+2094805 N

OT10 GGAGGTGGAACCCAGCCACT AAGAA 0.5 chr10:+121245867 N

OT11 GTGTCTGGTACACAGCCACA GAGAG 0.4 chr1:-4082085 N OT12 GGCTATGGTAGCCAGCCACT TTGGA 0.4 chr5:-56423952 N OT13 GGCTGTCGTCCGGAGCCACT CCGGG 0.4 chr7:+94656491 Y OT14 GGGAGTGATATGCAGCAACT TGGAG 0.4 chr7:-131250864 N OT15 GGGAGTGGTGGGCAGCTACT GTGAA 0.3 chr14:-44911835 Y OT16 GGGTCCAGTAGGCAGCCACT GTGGA 0.3 chr12:-123066785 N OT17 GGGTTAGGTCCGCAGCCAGT GCGGA 0.3 chr14:+70821992 N OT18 GTGTGTTGGACGCAGCCATT TTGAA 0.3 chr20:+8287936 N OT19 GGTTCTGGAACGCAGCCGCT GAGGG 0.2 chr7:-4862013 N OT20 GGGCGTGGTGGGCACCCACT GAGGA 0.2 chr15:-25213071 N OT21 GGGTAGGGTGCGCAGCCCCT TGGGG 0.2 chr1:+17827103 N OT22 GGGTGTGGCCCTCAGCCACG TTGAA 0.2 chr7:-4996920 N OT23 ATGTGTGGTACCCAGGCACT CTGAA 0.2 chr12:+24549803 N OT24 CGGTGAGGTATGCACCCACT GAGAA 0.2 chr7:+128850889 Y OT25 GGGTGTGGCACGCAGCCCCC GAGGG 0.2 chr7:+134729513 N OT26 GGGTATGCTAGGCAGGCACT GGGAA 0.2 chrX:22627670 N OT27 GGGGGTGGTGCGCAGCCTCA GAGGG 0.2 chr7:+63926722 N

OT28 GGCTGTGGGAAGCACCCACT GGGGG 0.2

chr10:+126920076 N

OT29 GGGTGGGGTCTGCATCCACT CTGGG 0.1 chr19:-2080122 N OT30 GGGTGTGGTACGCAGCAAAG CCGGG 0.1 chr1:+18854932 Y OT31 GGCTGTGGCACTCAGCCCCT TTGAA 0.1 chrX:+103928496 N OT32 GGCTGTGGTATGCAACAACT CTGGG 0.1 chr20:+12927432 N OT33 GGGTGTGTTACAGAGCCATT TTGAA 0.1 chr1:-93619830 N OT34 GGCTGTGGTAGGCAGCCTCC CTGGG 0.1 chr1:+26831667 N

OT35 GGGTGGGGGGCGCAGGCACT GAGGA 0.1 chr20:+62587869 N

OT36 CGGTGTGGTATGCAGACACC TAGGG 0.1 chr18:-30917069 N OT37 GGGTGCAGTAGGCAGGCACT GAGGA 0.1 chr1:-27381427 N OT38 GGGTGTGGGAAGGATCCACT GTGGG 0.1 chr3:+45895031 N OT39 GGGTGAGGCACACAGTCACT GAGAG 0.1 chr21:-37763578 N OT40 GGGTGTGGTTGGAAGGCACT CAGGG 0.0 chr5:-17056246 N OT41 GGATGTGGTACGTGGCCACG AAGGA 0.0 chr5:+156948122 N

OT42 GGGTGGAGTACGAAGACACT GGGGG 0.0 chr9:-73868952 N

OT43 GGGTGCGGTACACAGCCCCA CTGAG 0.0 chr5:+862965 N OT44 GGGTGAGGTAAGCAGCTCCT GAGAA 0.0 chr14:-80004704 N OT45 GGGGGTGGTACGCAGGCAGC ATGAG 0.0 chr15:+73696297 N OT46 GGGTGTGGGATGGAGCCCCT GGGAA 0.0 chr7:+155429802 N OT47 GGTTGTGGTACGGAGGCATT TAGGA 0.0 chr8:+61287911 N OT48 GGGTGTGATACCCAGACCCT GGGAG 0.0 chr10:+97875819 N OT49 GGGTATGGTAAGCTGTCACT GAGGG 0.0 chr14:+77594990 N