www.sciencemag.org/cgi/content/full/science.aas9129/DC1

Supplementary Materials for

Engineered CRISPR-Cas9 nuclease with expanded targeting space Hiroshi Nishimasu*, Xi Shi, Soh Ishiguro, Linyi Gao, Seiichi Hirano, Sae Okazaki,

Taichi Noda, Omar O. Abudayyeh, Jonathan S. Gootenberg, Hideto Mori, Seiya Oura, Benjamin Holmes, Mamoru Tanaka, Motoaki Seki, Hisato Hirano, Hiroyuki Aburatani,

Ryuichiro Ishitani, Masahito Ikawa, Nozomu Yachie, Feng Zhang, Osamu Nureki*

*Corresponding author. Email: nisimasu@bs.s.u-tokyo.ac.jp (H.N.); nureki@bs.s.u-tokyo.ac.jp (O.N.)

Published 30 August 2018 on Science First Release DOI: 10.1126/science.aas9129

This PDF file includes: Materials and Methods

Figs. S1 to S14

Tables S1 to S3

References

2

Materials and Methods

Sample preparationThe gene encoding SpCas9 (residues 1–1,368) was cloned between the NdeI and XhoI sites of the modified pET-28a vector (Novagen). The mutations were introduced by a PCR-based method, and the sequences were confirmed by DNA sequencing.

For in vitro cleavage experiments, the N-terminally His6-tagged SpCas9 proteins were expressed in Escherichia coli Rosetta2 (DE3) (Novagen). The SpCas9-expressing E. coli Rosetta2 (DE3) cells were cultured at 37°C in LB medium (containing 20 mg/l kanamycin) until the OD600 reached 0.8, and protein expression was then induced by the addition of 0.1 mM isopropyl β-D-thiogalactopyranoside (Nacalai Tesque). The E. coli cells were further cultured at 20°C overnight, and harvested by centrifugation. The E. coli cells were resuspended in buffer A (50 mM Tris-HCl, pH 8.0, 20 mM imidazole and 1 M NaCl), lysed by sonication, and then centrifuged. The supernatant was mixed with 1 ml Ni-NTA Superflow (QIAGEN), and the mixture was loaded into a Poly-Prep Column (Bio-Rad). The protein was eluted with buffer B (50 mM Tris-HCl, pH 8.0, 0.3 M imidazole and 0.3 M NaCl). The protein was loaded onto a 1 ml HiTrap SP HP column (GE Healthcare) equilibrated with buffer C (20 mM Tris-HCl, pH 8.0, and 0.3 M NaCl). The protein was eluted with a linear gradient of 0.3–1 M NaCl. The purified proteins were stored at −80°C until use. The 98-nt sgRNA (containing stem loops 1, 2 and 3) was transcribed in vitro with T7 RNA polymerase, and was purified with an RNeasy kit (QIAGEN). Staphylococcus aureus Cas9 (SaCas9) and its sgRNA were prepared as described previously (17). Acidaminococcus sp. BV3L6 Cas12a (AsCas12a), Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a), and their crRNAs were prepared as described previously (18).

For crystallization, the inactivating mutation (N863A) in the HNH domain was introduced into SpCas9-NG. The SpCas9-NG (N863A) protein was expressed in E. coli Rosetta2 (DE3), and was then purified using Ni-NTA and HiTrap SP HP columns. To remove the His6-tag, the purified protein was mixed with TEV protease, and was dialyzed at 4°C overnight against buffer D (20 mM Tris-HCl, pH 8.0, 40 mM imidazole, 0.3 M NaCl). The protein was passed through the Ni-NTA column equilibrated with buffer D. The protein was further purified by chromatography on a HiLoad Superdex 200 16/60 column (GE Healthcare) equilibrated with buffer E (10 mM Tris-HCl, pH 8.0, 150 mM NaCl and 1 mM DTT). The 81-nt sgRNA (containing stem loops 1 and 2) was transcribed in vitro with T7 RNA polymerase, and was purified by 10% denaturing (7 M urea) polyacrylamide gel electrophoresis.

3

Structure determinationSpCas9-NG was crystallized using a protocol similar to that for the SpCas9 VQR variant (14). The SpCas9-NG–sgRNA–DNA complex was reconstituted by mixing the SpCas9-NG protein, the 81-nt sgRNA, the 28-nt target DNA strand (Sigma-Aldrich) and the 8-nt non-target DNA strand (Sigma-Aldrich) (molar ratio, 1:1.5:2.3:2.7). The SpCas9-NG–sgRNA–DNA complex was purified by gel filtration chromatography on a Superdex 200 Increase 10/300 column (GE Healthcare) equilibrated with buffer F (20 mM HEPES-NaOH, pH 7.5, 250 mM KCl, 5 mM MgCl2 and 1 mM DTT). The purified SpCas9-NG–sgRNA–DNA complex was crystallized at 20°C, by the hanging-drop vapor diffusion method. The crystals were obtained by mixing 1 μl of complex solution (A260 nm = 15) and 1 μl of reservoir solution (0.1 M Tris-acetate, pH 8.0, 0.4 M KSCN and 14–16% PEG 3,350). The crystals were improved by microseeding, using Seed Bead (Hampton Research). The crystals were cryoprotected in buffer (0.1 M Tris-acetate, pH 8.0, 0.4 M KSCN, 30% PEG 3,350 and 10% ethylene glycol). The X-ray diffraction data were collected at 100 K on beamline BL41XU at SPring-8 (Hyogo, Japan), and were processed using XDS (19) and AIMLESS (20). The structure was determined by molecular replacement with MOLREP (21), using the SpCas9 structure (PDB: 4UN3) as the search model. The structure models were built using COOT (22) and refined using PHENIX (23). Molecular graphic images were prepared using CueMol (http://www.cuemol.org).

In vitro cleavage experimentThe BamHI-linearized pUC119 plasmid, containing the 20-nt target sequence and the PAM, was used as the substrate for in vitro cleavage experiments. The linearized plasmid target bearing the TGG PAM (100 ng, 5 nM) was incubated at 37°C for 30 min with the SpCas9–sgRNA complex (200 nM, molar ratio, 1:2), in 10 μl of reaction buffer, containing 20 mM HEPES-NaOH, pH 7.5, 100 mM KCl, 2 mM MgCl2, 1 mM DTT and 5% glycerol. The reaction was stopped by the addition of quench buffer, containing EDTA (20 mM final concentration) and Proteinase K (10 μg). The reaction products were analyzed using a MultiNA microchip electrophoresis system (SHIMADZU). To monitor the DNA cleavage time courses, the linearized plasmid target bearing the PAM (300 ng, 5 nM) was incubated at 37°C with the SpCas9–sgRNA complex (50 nM, molar ratio, 1:2), in 30 μl of reaction buffer. Aliquots (5 μl) were taken at 0.5, 1, 2 and 5 min, and mixed with 15 μl of quench buffer. The reaction products were analyzed using a MultiNA microchip electrophoresis system. In vitro cleavage experiments were performed at least three times, and representative electropherograms are shown.

The in vitro cleavage activity of SaCas9 was examined, using the pUC119 plasmid containing the 21-nt target sequence and the TTGAAT PAM. The in vitro cleavage activities of AsCas12a and LbCas12a were examined, using the pUC119 plasmid containing the 24-nt target sequence and the TTTA PAM.

4

PAM identification assayThe PAM identification assay was performed as described previously (10). The PAM library (80 ng) was incubated at 37°C with the purified SpCas9 (WT SpCas9 or SpCas9-NG) (100 nM) and the sgRNA (750 ng), in 10 μl of 1×CutSmart buffer (NEB). The reactions were quenched at the indicated times (<0.1, 1, 5, 15, 30 and 60 min) by the addition of 50 μl Buffer PB (QIAGEN), and were column-purified. The purified DNA was amplified with two rounds of PCR over 24 total cycles, using custom primers containing Illumina adaptors, and sequenced with a 75-cycle NextSeq kit (Illumina).

Indel analysisThe indel analysis was performed as previously described (14). Briefly, HEK293FT cells were plated one day prior to transfection in 96-well plates (Corning), at a density of 2.5 × 104 cells per well. Cells were transfected with the Cas9 plasmid (75 ng) and the sgRNA plasmid (25 ng) per well, using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s recommended protocol. Cells were harvested approximately 3 days post-transfection. Genomic DNA was extracted using a QuickExtract DNA extraction kit (Epicentre). PCR fragments for targeted deep sequencing were generated in two step PCR reactions, as previously described (24). Briefly, genomic regions of interest were amplified using primers with PCR handles for the second round of amplification, and then Illumina P5 adapters as well as unique sample-specific barcodes were attached to the first round PCR products.

GUIDE-seqThe human embryonic kidney (HEK) cell line 293FT (Fisher Scientific) was maintained in Dulbecco’ s modified Eagle’ s medium (DMEM, Life Technologies), supplemented with 10% fetal bovine serum (Gibco), at 37°C in a 5% CO2 atmosphere. GUIDE-seq experiments were

performed essentially as previously described (13). Briefly, 2 × 105 HEK293FT cells were transfected using a Lonza 4D-Nucleofector X Unit (program CM-137), in 20 μl Solution SE with 0.3 μg Cas9 and 0.2 μg sgRNA plasmids (1:1 molar ratio) targeting the EMX1 or VEGFA locus, along with 10 pmol of a GUIDE-seq end-protected dsODN containing an NdeI restriction site. The GUIDE-seq tag integration frequencies at the intended on-target sites were estimated by restriction-fragment length polymorphism (RFLP) assays, using the primers: 5′-CCATCCCCTTCTGTGAATGT-3′ (Fw) and 5′-GGAGATTGGAGACACGGAGA-3′ (Rev) for EMX1, and 5′-ACTGACTAACCCCGGAACCA-3′ (Fw) and 5′-AAATTACCCATCCGCCCCC-3′ (Rev) for VEGFA. Tag-specific amplification and library preparation were performed before high-throughput sequencing, using an Illumina NextSeq 500/550 High output V2 kit (300 cycles). GUIDE-seq data were analyzed with the GUIDE-seq analysis software (http://www.jounglab.org/guideseq).

5

Base-editing analysisHEK293Ta cells were maintained in DMEM (Sigma) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% Penicillin-Streptomycin (Sigma), at 37°C in a 5% CO2 atmosphere. 5 × 105 HEK293Ta cells/well were transfected with 300 ng of base-editor

plasmid and 100 ng of sgRNA plasmid, using 1.2 μl of 1 mg/ml Polyethylenimine (Polyscience). The cells were harvested 3 days after transfection, treated with 200 μl of 50 mM NaOH, incubated at 95°C for 10 min, and then neutralized with 20 μl of 1 M Tris-HCl, pH 8.0. Briefly, two round PCRs were performed for library preparation for high-throughput amplicon sequencing. Genomic regions of interest were first amplified to add the custom adapter sequence. The first PCR products were subjected to the second round PCR to attach Illumina TruSeq adapters and sample specific indices. After quantification using a KAPA Library Quantification Kit Illumina (KAPA Biosystems), the sequencing library was subjected to paired-end sequencing (600 cycles) on MiSeq (Illumina). The sequencing reads were demultiplexed based on sample specific indices and primer sequences and mapped to the target regions, using NCBI BLAST+ (version 2.6.0) with the blastn-short option. The C-to-T editing frequency and mutational spectrum for each target tested were normalized by subtracting the sequencing control samples (null vector transfection experiments), as described previously (16).

RuvC

HNH

PIPAM (5′-NGG-3′)

REC

5′NTS

TSDNA

SpCas9

3′

3′

5′

5′RNA

6

Fig. S1. Schematic of RNA-guided DNA cleavage by SpCas9.The 20-nt target sequence (protospacer) and the 5′-NGG-3′ PAM sequence (in the non-target strand) are colored blue and yellow, respectively. The sites cleaved by the HNH and RuvC nuclease domains are indicated by magenta and cyan triangles. TS, target strand; NTS, non-target strand.

R1333A1322

G1218

E1219 T1337

R1335

L1111

D1135

R1333A1322

G1218

E1219 T1337

R1335

L1111

D1135

NTS

TS

HNH

RuvC

REC

PI

RNA

PAM

GNG

7

Fig. S2. PAM recognition by SpCas9.In the crystal structure of SpCas9 in complex with the sgRNA and its target DNA (PDB: 4UN3), the second and third G nucleobases in the PAM are recognized by Arg1333 and Arg1335 in the PAM-interacting (PI) domain, respectively (7). TS, target strand; NTS, non-target strand.

E1219F

R1333

R1335V

E1219F

dG2*

1. WT SpCas9

2. R1335A

3. R1335A/L1111R

4. R1335A/G1218R

5. R1335A/A1322R

6. R1335A/T1337R

7. R1335A/L1111R/G1218R/A1322R/T1337R (ARRRR)

8. R1335A/L1111R/D1135V/G1218R/A1322R/T1337R (ARVRRR)

9. R1335A/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R (ARVRFRR)

0.5

–

3.0(kb)

2.01.0

UncleavedCleaved

WT

SpC

as9

R13

35A

R13

35A

/L11

11R

AR

RR

R

R13

35A

/T13

37R

R13

35A

/A13

22R

R13

35A

/G12

18R

WT SpCas9R1335A

R1335A/L1111R

ARRRRR1335A/T1337RR1335A/A1322RR1335A/G1218R

100806040200Cleavage (%)

(kDa)250

1 2 3 4 5 6 7 8 9

15010075

50

37

2520

1510

A

B C

F

D

E

3.0(kb)

2.0

0.5

1.0UncleavedCleaved

Time (min)WT SpCas9

0 0.5 1 2 5ARVRFRR

0.5 1 2 5

SpCas9-NG(VRVRFRR)

0.5 1 2 5ARVRRR

0.5 1 2 5ARRRR

0.5 1 2 5

0 1 2 3 4 5Time (min)

0

20

40

60

80

100

Cle

avag

e (%

)

WT SpCas9

ARRRRARVRRRARVRFRRSpCas9-NG (VRVRFRR)

8

Fig. S3. Engineering of SpCas9-NG.

9

(A) SDS-PAGE analysis of the wild-type (WT) and mutant SpCas9 proteins used for in vitro cleavage experiments. The gel was stained with SimplyBlue SafeStain.(B) In vitro DNA cleavage activities of WT SpCas9 and the R1335A, R1335A/L1111R, R1335A/G1218R, R1335A/A1322R, R1335A/T1337R and R1335A/L1111R/G1218R/A1322R/T1337R (ARRRR) mutants. A linearized plasmid target bearing the TGG PAM was incubated with the SpCas9–sgRNA complex (200 nM) at 37°C for 30 min, and the reaction products were then analyzed using a MultiNA microchip electrophoresis system.(C) Quantification of the DNA cleavage activities in (B). Data are shown as mean ± s.d. (n = 3).(D) In vitro DNA cleavage time courses of WT SpCas9 and the R1335A/L1111R/G1218R/A1322R/T1337R (ARRRR), R1335A/L1111R/D1135V/G1218R/A1322R/T1337R (ARVRRR), R1335A/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R (ARVRFRR), and R1335V/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R (VRVRFRR, SpCas9-NG) mutants. A linearized plasmid target bearing the TGG PAM was incubated with the SpCas9–sgRNA complex (50 nM) at 37°C for the indicated time points (0.5, 1, 2 and 5 min), and the reaction products were then analyzed using a MultiNA microchip electrophoresis system.(E) Quantification of the DNA cleavage time courses in (D). Data are shown as mean ± s.d. (n = 3).(F) Modeling of Phe1219 (E1219F) and Val1335 (R1335V) into the SpCas9–sgRNA–target DNA complex structure (PDB: 4UN3).

3.0(kb)

2.0

0.5

1.0

UncleavedCleaved

Time (min)PAM

WT SpCas9 (50 nM)

TGA0 0.5 1 2 5

TGT0 0.5 1 2 5

TGG0 0.5 1 2 5

TGC0 0.5 1 2 5

A

B

0.5

3.0(kb)

2.01.0

UncleavedCleaved

Time (min)PAM

SpCas9-NG (50 nM)

TGA0 0.5 1 2 5

TGT0 0.5 1 2 5

TGG0 0.5 1 2 5

TGC0 0.5 1 2 5

10

Fig. S4. In vitro DNA cleavage activities of WT SpCas9 and SpCas9-NG.(A and B) In vitro DNA cleavage time courses of WT SpCas9 (A) and SpCas9-NG (B). A linearized plasmid target bearing the TGN PAM was incubated with the SpCas9–sgRNA complex (50 nM) at 37°C for the indicated time points (0.5, 1, 2 and 5 min), and the reaction products were then analyzed using a MultiNA microchip electrophoresis system.

B

C

A

0 1 2 3 4 5Time (min)

0

20

40

60

80

100

Cle

avag

e (%

)

WT SpCas9

SpCas9-NGSaCas9

AsCas12aLbCas12a

3.0(kb)

2.0

0.5

1.0

UncleavedCleaved

Time (min)SaCas9

0 0.5 1 2 5AsCas12a

0 0.5 1 2 5LbCas12a

0 0.5 1 2 5

(kDa)

250S

aCas

9A

sCas

12a

LbC

as12

a

15010075

50

37

2520

1510

11

Fig. S5. Comparison of the in vitro DNA cleavage activities of WT SpCas9, SpCas9-NG, SaCas9, AsCas12a and LbCas12a.(A) SDS-PAGE analysis of SaCas9, AsCas12a and LbCas12a.(B) In vitro DNA cleavage time courses of SaCas9, AsCas12a and LbCas12a. A linearized plasmid target bearing the TTGAAT PAM was incubated with the SaCas9–sgRNA complex (50 nM) at 37°C for the indicated time points (0.5, 1, 2 and 5 min). A linearized plasmid target bearing the TTTA PAM was incubated with the Cas12a–crRNA complex (50 nM) at 37°C for the indicated time points (0.5, 1, 2 and 5 min). The reaction products were then analyzed using a MultiNA microchip electrophoresis system.(C) Quantification of the DNA cleavage activities in (B). Data are shown as mean ± s.d. (n = 3). The DNA cleavage time courses of WT SpCas9 and SpCas9-NG for the TGG PAM targets (Fig. 1, B and C) are shown for comparison.

12

Fig. S6. PAM identification analysis.(A) Histograms of the abundances of 48 PAMs (NNNNNNNN) at each in vitro cleavage time point for WT SpCas9 and SpCas9-NG. The color of each histogram represents elapsed time. NGG (WT SpCas9) and NGN (SpCas9-NG) sequences are shown in black.(B and C) PAM specificities of WT SpCas9 (B) and SpCas9-NG (C).

A

C

0 min

0 1 ≥2 0 1 ≥2 0 1 ≥2 0 1 ≥2 0 1 ≥2 0 1 ≥2

0

2000

4000

Cou

nt

WT

SpC

as9

SpC

as9-

NG

1 min 5 min 15 min 30 min 60 min

0

2000

4000

Cou

nt

Relative abundance

App

aren

t cle

avag

e ra

te c

onst

ant (

min

−1)

SpCas9-NG

0

0.05

0.1

A

C

G

T

C G TA

B WT SpCas9

0

1

2

≥3

App

aren

t cle

avag

e ra

te c

onst

ant (

min

−1)

A

C

G

T

C G TA

0.0

1.0

2.0

Bits

5 6 7 81 2 3 4PAM position

GG5 6 7 81 2 3 4

PAM position

0.0

1.0

2.0

Bits

AGT

13

A

TAATATTAGTAC

B

0 1 2 3 4 5Time (min)

0

20

40

60

80

100

Cle

avag

e (%

)3.0(kb)

2.0

0.51.0

UncleavedCleaved

Time (min)PAM

SpCas9-NG (50 nM)

TAA0 0.5 1 2 5

TAT0 0.5 1 2 5

TAG0 0.5 1 2 5

TAC0 0.5 1 2 5

Fig. S7. In vitro DNA cleavage activity of SpCas9-NG toward the TAN PAM targets.(A) In vitro DNA cleavage time courses of SpCas9-NG toward the TAN PAM targets. A linearized plasmid target bearing the TAN PAM was incubated with the SpCas9–sgRNA complex (50 nM) at 37°C for the indicated time points (0.5, 1, 2 and 5 min), and the reaction products were then analyzed using a MultiNA microchip electrophoresis system. Data are shown as mean ± s.d. (n = 3).(B) Quantification of the DNA cleavage activities in (A). Data are shown as mean ± s.d. (n = 3).

14

Arg1333Glu1219

Arg1335

E480K

E543D

M694I

R324L

E1219V

A262TS409I

G2 G3

HNH

RuvC

REC

PI

RNA

DNA

180º

Fig. S8. xCas9 mutations.The seven mutations in xCas9 (xCas9 3.7) are mapped on the SpCas9–sgRNA–target DNA complex structure (PDB: 4UN3). SpCas9-NG and xCas9 do not share the identical mutation. E1219V and A262T/R324L/S409I/E480K/E543D/M694I are located in the PI and REC domains, respectively. Glu1219 forms salt bridges with Arg1335, suggesting that the E1219V mutation increases the flexibility of Arg1335 to allow the less stringent recognition of the third PAM nucleotide. Ala262, Arg324 and Ser409 are located near the cleavage site in the target DNA strand (magenta circle), suggesting that the A262T/R324L/S409I mutations may facilitate the DNA cleavage by the HNH domain. Met694 forms hydrophobic interactions with the RNA–DNA heteroduplex, suggesting that the M694I mutation could affect the interactions with the RNA–DNA heteroduplex. The roles of the E480K and E543D mutations are unclear, since Glu480 and Glu543 are located farther away from the nucleic acids and exposed to the solvent.

15

E F GWT SpCas9 (200 nM) xCas9 (200 nM)SpCas9-NG (200 nM)

0 5 10 15 20 25 300

20

40

60

80

100

Time (min)

Cle

avag

e (%

)

0 5 10 15 20 25 300

20

40

60

80

100

Time (min)

Cle

avag

e (%

)

0 5 10 15 20 25 300

20

40

60

80

100

Time (min)

Cle

avag

e (%

)

3.0(kb)

2.0

0.5

1.0

UncleavedCleaved

Time (min)PAM

xCas9 (50 nM)

TGA0 0.5 1 2 5

TGT0 0.5 1 2 5

TGG0 0.5 1 2 5

TGC0 0.5 1 2 5

A

3.0(kb)

2.0

0.5

1.0

UncleavedCleaved

Time (min)PAM

WT SpCas9 (200 nM)

TGA0 2 5 15 30

TGT0 2 5 15 30

TGG0 2 5 15 30

TGC0 2 5 15 30

B

3.0(kb)

2.0

0.5

1.0

UncleavedCleaved

Time (min)PAM

xCas9 (200 nM)

TGA0 2 5 15 30

TGT0 2 5 15 30

TGG0 2 5 15 30

TGC0 2 5 15 30

D

C

3.0(kb)

2.0

0.5

1.0

UncleavedCleaved

Time (min)PAM

SpCas9-NG (200 nM)

TGA0 2 5 15 30

TGT0 2 5 15 30

TGG0 2 5 15 30

TGC0 2 5 15 30

TGATGTTGGTGC

Fig. S9. Comparison of the in vitro DNA cleavage activities of WT SpCas9, SpCas9-NG and xCas9.(A to D) In vitro DNA cleavage time courses of xCas9 (50 nM) (A), WT SpCas9 (200 nM) (B), SpCas9-NG (200 nM) (C) and xCas9 (200 nM) (D). A linearized plasmid target bearing the TGN PAM was incubated with the SpCas9–sgRNA complex at 37°C for the indicated time points, and the reaction products were then analyzed using a MultiNA microchip electrophoresis system.(E to G) Quantification of the DNA cleavage activities in (B to D). Data are shown as mean ± s.d. (n = 3).

16

Fig. S10. Crystal structure of SpCas9-NG.(A) 2mFO − DFC electron density maps for the key residues (contoured at 1.0σ) and the non-target DNA strand (contoured at 2.2σ) (stereo view).(B) Structural comparison of SpCas9-NG with the SpCas9 R-loop complex (PDB: 5F9R) (semi-transparent blue).

NTS

TS

A1322R R1333

SpCas9-NGSpCas9 R-loop (PDB: 5F9R)

R1335V

E1219F

G1218R

T1337R

D1135V

L1111R

-2* -1* 1*2*

3*

E1219FR1333

R1335VT1337R

dT1* dG2* dG3*

E1219FR1333

R1335VT1337R

dT1* dG2* dG3*A

B

17

Fig. S11. Off-target sites identified by GUIDE-seq.GUIDE-seq on-target and off-target reads for the EMX1 and VEGFA sites are shown for SpCas9, SpCas9-ES, SpCas9-NG and SpCas9-NG-ES. The on-target sequences are indicated by open squares. The total numbers of identified off-target sites are shown in parentheses. The top 50 off-target sites are shown for the VEGFA target for SpCas9 and SpCas9-NG.

SpCas9-NG – VEGFA (257)1 10 20G G T G A G T G A G T G T G T G C G T G N G G Reads

A • • • • • • • T • • • • • • • T • • • • • T 9520A • • • • • • • • • • • • • • • T • • • • • T 8713T • • • • • • • • • • • • • • • T • • • • • T 3380• • • • • • • • • • • • • • • • T • • • • • T 2038• A • • • • • • T • • • • • • • T • • • • • T 1254T • • • • • • • • • • • • • • • T • • • • • A 904T • • • G • • • • • • • • • • • • • • • • • A 684C A G • • • • • • • • • • • • • • • • • • • T 680• • • • • • • • T • • • • • • • T • • • • • T 665A • • • • • A • • • • • • • • • T • • • • • T 665T A • • • • • • • • • • • • • • T • • • • • T 619• • • • T • • • • • • • • • • • T • • • • • A 615• • • • T • • • • • • • • • • • • • • • • • T 566A • • • • • • • • • • • • • A • • • • • • • T 542A • C • • • • • G • • • • • • • • • • • • • • 531• • • • T • • • • • • • • • • • T • • • • • T 490• • • • • • • • • • • • • • • • • • • • • • • 435A • • T • • • • • • • • • • • • • • • • • • T 369T • • • • • • • • • • • • • • • • • • • • • A 360A • • • • A • A • • • • • • • • • • • • • • T 357T • • • • • • • • • • • • • • • • A • • • • A 329C A • • • • • • • • • • • • • • T • • • • • T 327C • • • • • • • C • • • C • • • • • • • • • T 316A • • • • • • • • • • • • • • • • A • • • A T 316A • • • • A • • • • • • • • • • T • • • • • T 313A A G • • • • • • • • • • • • • A • • • • • T 299A • • • • A • • • • • • • • • • • A • • • • A 298A • • • • • • • • • • • • • • • T • • • • • • 293T • • • • • • • T • • • • • • • T • • • • • T 286A • • • • • • • • • • • • • • • T • • • • • T 277• A • • • • • • • • • • • • • • A • • • • • A 270T • • • • • • • T • • • • • • • • • • • • • T 259• • • • • • • • T • • • • • • • A • • • • A • 251• • A • • • G • • • • • • • • • • • • • • A T 246• A • • • • • • T • • • • • • • T • • • • • • 244C C • • • • • • • • • • • • • • T • • • • • T 219A • • • • • • • • • • • • • • • • • C • • • C 217T A • • • • • • • • • • • • • • • A • • • • T 216T • • • G • • • • • • • • • • • • • • • • • • 215A • • • T • • • • • • • • • • • • • • • • • T 211C • • • • • • • • • • • A • • • A • • • • • T 170T • • • • • • • G • • • • • • • • • • • • • T 169A C • • • • • • • • • • • • • • T • • • • • T 162T • • • • • • • T • • • • • • • • • • • • • C 160T • • • • • • • • • • • • • • • T • • • • • C 159A A • • • • • • C • • • • • • • T • • • • • T 152A • • • T • • • • • • • • • • • T • • • • • T 151• • • • • A • • • • • • • • • • T • • • • • T 149T • • • • • • • • • • • • • • • A • • • • • T 146A • • • • • • • G • • • • • • • • • • • • • T 143• • • • T • • • • • • • • • • • T • • • • • T 138

SpCas9-ES – EMX1 (2)1 10 20G A G T C C G A G C A G A A G A A G A A N G G Reads• • • • T A • • • • • • • • • • • • • • • • • 2450• • • • • • • • • • • • • • • • • • • • • • • 1991• • • G • • • • • • • • • • • • • A G • • • • 1124

SpCas9-NG – EMX1 (17)1 10 20G A G T C C G A G C A G A A G A A G A A N G G Reads• • • • • T A • • • • • • • • • • • • • • A • 4502• • • • • • • • • • • • • • • • • • • • • • • 3355• • • • • • T • • • • • G • • • • • • • • A • 1978• • • • T A • • • • • • • • • • • • • • • • • 318• • • • A G • • • • • • G • • • • • • • • • A 315C • • • • • C • • • • • • • • • G A • G • T • 232• • A • • • A • • • • • G • • • • • • • • • A 191• • • • • A • • • • • • • • • • • T G C • C T 130• • • • T • A • • • • • • G A • G A • • • • • 101• • • • • A • • • • • • • • • • • A G • • • A 68A G • • • T • • • • • • • • • • • A G • • • • 67• • • C G • • • • • • • • • • • • A • G • A • 65• • • G • T • • • • • • • • • • • • • • • A C 44• • • • • • • G • A • • G • • • • • • • • • • 42• T • • • T • • • • • • • G • • • • • • • • A 38A • • G • A • • • • • • • • • • • • • G • • T 21• • • • • A • G • • • • • • • • • • • • • A T 20A C • • • T • • • • • • • • • • • • • • • • • 7

PAM

PAM

PAM

SpCas9-ES – VEGFA (13)1 10 20G G T G A G T G A G T G T G T G C G T G N G G Reads

A • • • • • • • • • • • • • • • T • • • • • • 2395• • • • • • • • • • • • • • • • • • • • • • • 1488• • • • • • • A • • • • • • A • • • • A • • • 467T • • • • • • • • • • • • • • • T • • • • • A 402A • • • • • • • • • • • • • • • T • • • • • A 389• • • • • • • • • • • • • • • • T • • • • • • 315A • • • • • • • • • • • • • A • T • C • • • • 120• • • • • • • • • • • • A • • • A • • • • • • 118• • • • • • • • • • A • • • • • T • • • • • • 108• C • • • • • • • • • • • A • • • • • • • • • 59• • • • • • • • • • • • C • • • • • G • • • • 38A • • • • • • • • • • • • • • • T • • • • • A 35T • • • • • • • • • • • • • • • • • • • • • A 22• • • • • • • C • • • • • • • • A • • • • • • 9

SpCas9-NG-ES – VEGFA (49)1 10 20G G T G A G T G A G T G T G T G C G T G N G G Reads

A • • • • • • • • • • • • • • • T • • • • • T 21061• • • • • • • • • • • • • • • • • • • • • • • 7624• • • • • • • • • • • • • • • • T • • • • • T 1272A • • • • • • • • • • • • • A • • • • • • • T 881A • • • • • • • • • • • • • • • T • • • • • • 752A • • • • • • • • • • • • • • • T • • • • • A 657T • • • • • • • • • • • • • • • T • • • • • T 399T • • • • • • • • • • • • • • • • • • • • • A 365A • • • • • • • T • • • • • • • T • • • • • T 259A • • • • • • • • • • • • • • • A • • • • • T 257A • • • • • • • • • • • A • • • • • • • • • T 254T • • • • • • • • • • • • • • • T • • • • • A 250• • • • • • • • • • • • • • • • A • • • • A T 227A • • • • • • • • • • • • • • • • A • • • A T 218A • • • • • • • • • • • • • • • T • • • • • T 195A • • • • • • • • • • • • • • • • • C • • • C 186C A G • • • • • • • • • • • • • • • • • • • T 177A • • • • • • • • • C • • • • • T • • • • • T 170T • A • • • • • • • • • • • • • T • • • • • T 159A • • • • • • • • • • • • • • • A • • • • • T 155T • • • T • • • • • • • • • • • A • • • • • A 135A • • • • • • • G • • • • • • • • • • • • • T 129T T • • T • • • T • • • • • • • • • • • • A T 113T • • • • • • • • • • • • • • • T • • • • • C 102A • • • • • • • • • • • • • • • T • • • • • A 94• A • • • • • • • • • • • • • • A • • • • • A 76• • A • • • • • • • • • • • • • T • • • • • T 73A • • • • • • • • • • A • • • • T • • • • • T 73A • • • • • • • • • • • • • • • T • • A • • T 69• • • • • A • • • • • • • • • • T • • • • • T 60A • • • • • • • • • • • A • • • • • • • • • T 60• • • • • • • • • • • • • • • • A • • • • • T 49• • • • • • • • • • • • • • • • T • • • • • • 47A • • • • A • • • • • • • • • • T • • • • • T 46• • • • • • • • • • • • G • • • • • • • • • T 45A • • • • • • • • • • • A • • • A • • • • • T 40• • • • • • • • T • • • • • • • T • • • • • T 32T • • • G • • • • • • • • • • • • • • • • • A 26T • • • • • • • • • C • • • • • • • • • • • T 25• • • • • • • • • • • • • • • • T • • A • • T 22T • • • • • • • • • • • • • • • A • • • • • T 17• • • • • • • • • • • • • • • • A • • • • • T 16A • • • • • • • • • • • • • • • T • • • • T T 14A • • • • • • • • • • • • • • • T • • • • A A 13• • • • • • G • • • • • • • • • T • • • • • T 10T • • • • • • • • • • • • • • • • A • A • • T 9• A • • • • • • • • • • • • • • T • • • • • T 8• • • • • • • A • • • • • • A • • • • A • • • 6T • • • • • • • • • • • • • • • • A • • • • A 5• • • • • • • • • • • • A • • • A • • • • • T 3

SpCas9-NG-ES – EMX1 (2)1 10 20G A G T C C G A G C A G A A G A A G A A N G G Reads• • • • • • • • • • • • • • • • • • • • • • • 4355• • • • • • T • • • • • G • • • • • • • • A • 31A G • A • G • • T • • • • C • • • • • G • • • 8

PAM

PAM

PAM

SpCas9 – VEGFA (95)1 10 20G G T G A G T G A G T G T G T G C G T G N G G Reads

T • • • G • • • • • • • • • • • • • • • • • • 828A • A • • • • • • • • • • • • • • A • • • • • 369• C • • • • • • • • • • • A • • • • • • • • • 326• • • • • • • • • • • • • • • • • • • • • • • 300A • • • • • • • • • • • • • • • T • • • • • • 245A • • • T • • • • • • • • • • • • • • • • • • 162• T • • • • • • • A • • • • • • • • • • • • • 158A • C • • • • • G • • • • • • • • • • • • • • 155T • • • • • • • • • • • • • • • T • • • • • A 145T • • • G • • • • • • • • • • • • • • • • • A 140• • • • • • • • • • • • C • • • • • G • • • • 139• A • • • • • • • • • • A • • • A • • • • • • 130T • • • • • • • • • • • • • • • • • • • • • A 120T • • • • • • A • • • • • • • • T • • • • • • 100• • • • • • • • T • • • • • • • • A • • • • • 97• • • • • • • • • • • • • • • • T • • • • • • 89A • • • • • • • • • • • A • • • A • • • • • • 75A C • • T • • • • • • • • • • • • • • • • • • 73• • • • T • • • • • • • • • • • • A • T • • • 70A • • • • A • • • • • • • • • • T • • • • • • 68C • • • • • • • • • • • • • • A • C • • • • • 67• • • • • • • C • • • • • • • • A • • • • • • 64• • G • • A • • • • • • • • • • • A • • • A • 59• • • • • • A • • • • • • • • • • • • A • • A 58• • • • • • • • • • • • A • • • A • • • • • • 57• T A • • • • • • • • • • • • • T • • • • • • 52A • G • • • • • • C • • • • • • • • • • • • • 51T • • • • • • • • • • • • • • • T A • • • • • 49A • • • • • • • • • • • • • • • T • • • • • A 48• • • • G • • • • A • • G • • • • • • • • • • 47• • • • T • • • • • • • • • • • T • • • • • • 46A • G • • • • • • • • • • • A • A • • • • • • 44• • • • • • • A • • • • • • A • • • • A • • • 41• • A • • • • • • • • • • • • • • A • • • • C 40• • • • T • • • • • • • • • • • T • • • • • A 39C • C • • • • • • • • • • • • • • • C • • • • 38A • • • • • • • • • • • • • • • T • • • • • T 38• A • • • • • • T • • • • • • • T • • • • • • 37• A • • • • • • • • • • • • • • A • • • • • A 35A • A • • • • • • • • • • • • • T • • T • • • 35T • • • G • • • • • • • • • • • T • • • • • • 34A • • • • • • A • • • • A • • • A • • • • • • 34• • • • • • A • • • • • • • • • • A C • • • • 31T • • • • • • • • • • • • • • • T • • • • A • 28A • C • • • • • • • • • • • • • T • • • • • • 26A • A • • • • • • • • • • • • • T • • • • • A 26A A • • • • • • • • • • • • • • A • • • • A • 26• • • • • • • • • • A • • • • • T • • • • • • 25• • • • • • C A • • • • • • • • T • • • • • • 25A • • • • • • A T • • • • • • • T • • • • • • 25T • • • • • • • G • • • • • • • • A • • • • • 24

SpCas9 – EMX1 (24)1 10 20G A G T C C G A G C A G A A G A A G A A N G G Reads

PAM

PAM

• • • • • • • • • • • • • • • • • • • • • • • 11537• • • • T A • • • • • • • • • • • • • • • • • 6726• • • • • T A • • • • • • • • • • • • • • A • 5502• • • G • • • • • • • • • • • • • A G • • • • 2065• • • • • • T • • • • • G • • • • • • • • A • 1634• • • C • G • • • • • • • • • • • • G • • • • 1352A • • • • T • • • • • C • • • • • • • • • • • 1174• • A • • • A • • • • • G • • • • • • • • • A 670• • • • • • • G • A • • G • • • • • • • • • • 652• • • G • • • G • • • • G • • • • • G • • • • 449A C • • • T • • • • • • • • • • • • • • • • • 304A • • • • A • • • • • • • • A • • • • G • • • 161• • • • A G • • • • • • G • • • • • • • • • A 159A • • • • • • • • G • • • G • • • • • • • • • 155A • • • • • C G • • • • • G • • • • • • • • • 139• • • • • T • G • • • • G • • • • • • • • A • 133• • • C • • • G • • • • G • • • • • • T • • • 77C • • • • A • G • • • • • G • • • • • • • • • 75• • • • • • C • • • • • • • • G • C G • • • • 66• • • • • • C • • • • A • • • • • • • • • A • 46• • • • • T A • • • • • G • • • • T • • • • • 40A • • • T G • • • • • • G • • • • • • • • • • 34A • • • • A • • • G • • • • • • • • • • • • • 29A • • C • • • • • • • • • • • • • • T T • A • 13• • A • • • A • • • • • • • • • • • • G • A • 7

18

Fig. S12. Base editing in human cells.(A) Base-editing spectra of Target-AID and Target-AID-NG at the 20 endogenous target sites.(B) Base-editing spectra of Target-AID, Target-AID-NG and xCas9-BE4 at the 12 poly-C sites. Relative mutated base occupancies are shown for positions with mutation frequencies of ≥1%.

A T G C

Protospacer PAM

Target-AID Target-AID-NG

EMX1 FANCF GRIN2B MECP2 VEGFAEMX1 FANCF GRIN2B MECP2 VEGFA

Relative distance from PAM (nt) Relative distance from PAM (nt)

Mut

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nfre

quen

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NG

A

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(%)

20 10 0 20 10 0 20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0 20 10 0 20 10 0

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NG

G

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20 10 0 20 10 0 20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0 20 10 0 20 10 0

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20 10 0 20 10 0 20 10 0 20 10 0 20 10 00

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NG

C

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20 10 0 20 10 0 20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0 20 10 0 20 10 0

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20 10 0 20 10 0 20 10 0 20 10 0 20 10 00

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NG

T

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(%)

20 10 0 20 10 0 20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0 20 10 0 20 10 0

0

10

20

30

20 10 0 20 10 0 20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0 20 10 0 20 10 00

25

50

75

100

A

19

NGA

PolyC-1 PolyC-2 PolyC-3

NGT

PolyC-1 PolyC-2 PolyC-3

NGG

PolyC-1 PolyC-2 PolyC-3

NGC

PolyC-1 PolyC-2 PolyC-3

A T G C

Protospacer PAM

Relative distance from PAM (nt)

0

10

20

30

20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 00

25

50

75

10020 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0

0

10

20

30

20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 00

25

50

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10020 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0

0

10

20

30

20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 00

25

50

75

10020 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0

20 10 0 20 10 0 20 10 0

Targ

et-A

ID Mut

atio

nfre

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(%)

Targ

et-A

ID-N

G

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(%)

xCas

9-B

E4 Mut

atio

nfre

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cy(%

)

Pro

duct

dist

ribut

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(%)

B

20

Fig. S13. C-to-T editing efficiencies of Target-AID and Target-AID-NG at the 32 endogenous target sites in HEK293T cells.

NG

A

NG

T

NG

G

NG

C

NG

A

NG

T

NG

G

NG

C

0

10

20

30

40

50C

-to-T

edi

ting

frequ

ency

(%)

Target-AID Target-AID-NG

21

WT SpCas9 (NGG)

VQR (NGA)

VRER (NGCG)

WT + VQR + VRER (NGG/NGA/NGCG)

SpCas9-NG (NG)

0

5

10

15

≥20

Human genome (non-masked) Human coding sequences (non-masked)

Dis

tanc

e to

nea

rest

cle

avag

e si

te (b

p)

Fig. S14. Targeting range of SpCas9-NG.Comparison of the targeting ranges of WT SpCas9, SpCas9-NG, and the previously described SpCas9 variants (VQR, VRER, and WT + VQR + VRER) in the human genome and coding sequences. Plots show the probability mass function of the distance (in base pairs) to the nearest cleavage site. The boxplots indicate median and interquartile range. Genomic regions that contain Ns or masked repeats were ignored in this analysis. SpCas9-NG increases the targeting range of SpCas9 to one cleavage site for every ~2.2 bp in the human coding sequence (~3.1-fold increase relative to WT SpCas9 alone). Importantly, SpCas9-NG can efficiently target NGT sites that are inaccessible by WT SpCas9 or the VQR and VRER variants (4).

22

Table S1 Data collection and refinement statistics.

Data collection SPring-8 BL41XU

1.000C2

177.4, 68.6, 188.090, 111.5, 90

0.099 (1.202)0.063 (0.778)

9.5 (1.7)100 (100)3.5 (3.4)

0.992 (0.553)

58,0870.218/0.244

10,5352,464

1348

89.085.187.551.7

0.0020.452

97.182.74

BeamlineWavelength (Å)Space groupCell dimensionsa, b, c (Å)α, β, γ (°)Resolution (Å)*RmergeRpim I/σI Completeness (%)MultiplicityCC(1/2)

RefinementResolution (Å)No. reflectionsRwork/RfreeNo. atoms

ProteinNucleic acidIonSolvent

B-factors (Å2)ProteinNucleic acidIonSolvent

R.m.s. deviationsBond lengths (Å)Bond angles (°)

Ramachandran plot (%)Favored regionAllowed regionOutlier region 0.08

*Values in parentheses are for the highest resolution shell.

48.17–2.70 (2.77–2.70)

48.17–2.70

23

PAMGGGTGGGGTGGTGGAAGAGGCAGCGGGGGGCGTGGTCGATGAAGCCGGTGTGGTCGAAGACGCCGGAGTGGTAGAAGAGGCGGCGGGAGGAGTGGTCGAGGACGCAGGTGGCGTAGAGGAGGCCGCGGGAGTGGTGGATGAGGCGGCCGGCGGCGTAGTCGAGGATGCCGCTGGAGGGGTGGATGGAGGGGTGGTGGAAGAAGCGGC

Site #121212121212121112121112121212121211211212112121212121212121112121212

Sequence (5′ to 3′)GGCCCCAGTGGCTGCTCTGGGTCACCTCCAATGACTAGGGGCAGGGATCCAAGCACACAAGGGCTCCCATCACATCAACCGCAGCCAGCGACGTGCCCCAGAGGACAAAGTACAAACGGCGTCTCTCTCTTAATGACACGGCTTTACCCAGTTCTCTGGGGATGTCTGCAGGCCAGATGAGCTGAGGTGCAGAATCCAGGGCTCTCAGGCCCTGTCCGCAGGTTCCGTGCCAGCTCCCAAGGGAAGCTGGGTGAATGGAGGCCACCACAGGGAAGCTGGGGCGCTCGGCCACCACAGGGAGTTGCCCTCATTATTCAGCAGTACCTATCTGAGCATACCGGTCAGTGGAGAGCACCATAAGCATCTCTGTGTATACCAAAGGACTGTTTGAAAGGTGCAGGCTCCAGCCTAATTTCCCAAGCAGGAAGAACAGTTCAAGAGCAAATACCAGAGATAAGAGGCTCTGTCAGAAGATCTCCAGCTGTAACAGGAGGGCCAGGGAGCAAATACCAGAGATAAGGCATTGCTGTCATCCTCGTGGATCAGGAAATAGAGCCACAGCAGAGCTAGGGGTTCAGAGGCCTGCATATTACAACCAAGGCCTTCATAGAATTGAAGAGGGAACCTGTGAGCTGCACCAGCGTACTTCGAAAAGGTAGGGGTCCTGGAGAAAAGTCCTGGTATCCGGAGAGAATTTGCAGGACTCTCTGATGAAGACCCGGAATCCCTTCTGCAGCACCGCGGTCTCAAGCACTACCTAGCGCTTCAATGGCTATAGAGGAGAACCCAAATCTCCAGGAGCGGCTGCACAACCAGTGGAGTGACGTCCTGCTCTCTCTGGGAGGCTATCAACAAAGAATGCAGATTTCTTGAGTCCAGGGTTTGCAGTTGGCTAAGAGAGTACCTAATGGACTTCAGGGGTACTCATACCAAAAAAGAGGTTCTGTCACCAACTGAAGTGCTACGCTAGCAGTGAGCAAGCCCGCTGCCGTATCCCGAGGAGCCCTCCCCGACCCACCGGCTCCTTTAACCTTGACGAAGTACGGTGTTGAATACCAGTGCGCTTCCCGCGGAGCACCTGGTTGCCCCCAACAGCCCCAGCAGAGGTAGAATCAGCAGGGGACCATGAGATAGAAGGCGGTAAACCTGGAATAACACGAGCCCAGTTCGAACTTCCTGGGCAAGGGCTGAGCAGCAGTAGATGGCCCCTAGAAAAGCAAGTACAGTTAGTACTCAGCAGGCAGTTGTAGGGAAATAGGGGCTTTGCTACAACCCCAGCAGCTTCGAAGTGCTTGAAAATGTAAGCATAGAGGTGCTATGGTAGACCAGGAGAATAAAGGGCCCATCCCTGAGTCCAGCGGCACTTGTCCGGGTAAGTTG

Locus

UBE3A

SHANK3

CUL3

EMX1

VEGFA

DYRK1A

GRIN2B

MECP2

PTEN

FANCF

Table S2 Target sequences of the indel analysis.

24

Table S3 Target sequences of the base-editing analysis.

Locus PAM Sequence (5′ to 3′)

GGA GAGGACAAAGTACAAACGGC

GGT GGGCTCCCATCACATCAACC

GGG GGCCCCAGTGGCTGCTCTGG

AGC GCTTTACCCAGTTCTCTGGG

TGA GCCACCACAGGGAAGCTGGG

CGT GCTCTCAGGCCCTGTCCGCA

GGG GATGTCTGCAGGCCAGATGA

AGC GCGCTCGGCCACCACAGGGA

AGA GAGCAAATACCAGAGATAAG

GGT GCTCTGTCAGAAGATCTCCA

GGG GTCTGACCGGAAGATCCAGG

GGC GATCAGGAAATAGAGCCACA

AGA GCGCTTCAATGGCTATAGAG

CGT GCGGTCTCAAGCACTACCTA

TGG GGAATCCCTTCTGCAGCACC

CGC GTGACGTCCTGCTCTCTCTG

CGA GCGTACTTCGAAAAGGTAGG

AGT GCCTTCATAGAATTGAAGAG

AGG GCCTGCATATTACAACCAAG

CGC GTATCCGGAGAGAATTTGCA

GGA CCCCCCACAGAACATATAGA

CGT CCCCCCACTGAATAGCACAT

GGG CCCCCCATCTCACCACCGCA

GGC CCCCCCAGAGAACAATACAA

GGA CCCCCCCATGAACATCCAAG

GGT CCCCCCATAGTGGTGACACG

GGG CCCCCCAGTGAAGTAATTAA

AGC CCCCCCACAGAATATCAAGG

GGA CCCCCCATCGAGGCGAGCAA

GGT CCCCCCATATCCTCTCAGGA

CGG CCCCCCACCTGCCTTCCACG

CGC CCCCCCATCATGTAGCCACA

EMX1

VEGFA

GRIN2B

FANCF

MECP2

PolyC-1

PolyC-2

PolyC-3

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