1. Figure S1. Effect of lipid-based transfection reagents on genome editing
Figure S1. Various amount of Cas9 protein and gRNA of HPRT were transfected into
293FT cells using Lipofectamine 2000, Lipofectamine 3000 or RNAiMAX. Upon 48 hours
post transfection, the cells were lysed for GCD assays.

2. Figure S2. Effect of dose on off-target mutagenesis
Figure S2. The HEK293FT cells were transfected with various amount of Cas9 protein while
keeping the ratio of Cas9/gRNA constant. The cells were lysed upon 48 hours post transfection. On target mutagenesis at the VEGFA target site 3 and off target mutagenesis at OT3-2 were quantified by DNA sequencing. 96 clones sequenced for each on- and off-target data point. All data from a single biological replicate.

3. (A) 24 optimized protocol
Figure S3. Optimization of electroporation using Neon 24 optimized protocol
(A) 24 optimized protocol
Protocol 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Pulse Voltage
1400
1500
1600
1700
1100
1200
1300
1000
850
950
1050
1150
Pulse Width 30 40
# of Pulse
(B) Transfection of Jurkat T cells via electroporation
protein
DNA
mRNA
Figure S3. A mastermix of plasmid DNA, mRNA or Cas9 protein/gRNA were prepared in 250 µl of Resuspension Buffer R and then mixed with 50 x 105 Jurkat T cells, which were previously washed with PBS. An aliquot of 10 µl sample was drawn with a Neon 10 µl pipette tip, followed by electroporation using Neon 24 optimization protocol, which varies in pulse voltage, pulse width and number of pulses (A). (B) The percentage of genome cleavage for each electroporation condition 1-24 and each delivery method (Cas9 protein- RNP complex, DNA - plasmid, or mRNA- mRNA/gRNA)was estimated 48-hour post transfection using the GCD assay. Arrows indicate the expected cleavage product.

4. (A) 24 optimized protocol (C) Flow cytometry
Figure S4
(A) 24 optimized protocol
(C) Flow cytometry
Unstained
Control
Tra AlexaFluor®488
Cas9 mRNA
Cas9 Protein
mRNA
Cas9
(B) Transfection via Electroporation
Neg DNA mRNA ug Cas9
SSEA4-AlexaFluor®647
% Cleavage:
Figure S4. Genome editing in human iPSC. (A) Cas9:gRNA complexes were delivered into iPSC using Neon 24 optimized protocol. (B) iPSC were transfected with plasmid DNA, mRNA and various amount of Cas9:gRNA complexes. The genome modification was determined by Genomic Cleavage assay. The percentage of cleavage was confirmed by DNA sequencing. (C) The transfected iPSC were double-stained with TRA-1-60 Alexa Fluor® 488 and SSEA4 Alexa Fluor®647 conjugated antibodies, followed by flow cytometric analysis.

5. Figure S5. Time course of Cas9 RNP turnover and cleavage analysis in Mouse ESCs
NC h
% Cleavage
(B)
Cas9
Actin
Figure S5. Mouse ESCs were transfected with Cas9 RNPs (1.5 ug Cas9 protein/300 ng gRNA) directed to the Rosa 26 locus. (A) Cell samples were taken at different time points and analyzed by GCD assays. Arrows indicate cleavage products. (B) Western blot analysis of samples taken at different time points.

6. Sequences used for gRNA synthesis are color coded to match Figure 2A.
Table S1. Oligonucleotide sequences used for gRNA synthesis and sequences of constructs used
Constant Forward
GTTTTAGAGCTAGAAATAGCAAG
Universal Forward
TAATACGACTCACTATAG
Universal Reverse
AAAAGCACCGACTCGGTGCCAC
Target F1-HPRT
TAATACGACTCACTATAGGCATTTCTCAGTCCTA
Target R1-HPRT
TTCTAGCTCTAAAACTGTTTAGGACTGAGAAATG
Target F1-AAVS1
TAATACGACTCACTATAGGCCAGTAGCCAGCCCC
Target R1-AAVS1
TTCTAGCTCTAAAACGGACGGGGCTGGCTACTGG
Target F1-RelA
TAATACGACTCACTATAGGAGGGGGAACAGTTCT
Target R1-RelA
TTCTAGCTCTAAAACTTTCAGAACTGTTCCCCCT
GCD F-HPRT
ACATCAGCAGCTGTTCTG
GCD R-HPRT
GGCTGAAAGGAGAGAACT
GCD F-AAVS1
GAATATGTCCCAGATAGCAC
GCD R-AAVS1
GTTCTCAGTGGCCACCCTGC
GCD F-RelA
AGTACAACAGGCCCTGATTC
GCD R-RelA
TCCTCTCGCCTGGGATGCTG
ct/tracr constant region
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT
Complete gRNA-HPRT
TAATACGACTCACTATAGGCATTTCTCAGTCCTAAACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT
Complete gRNA-AAVS1
TAATACGACTCACTATAGGCCAGTAGCCAGCCCCGTCCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT
Complete gRNA-RelA
TAATACGACTCACTATAGGAGGGGGAACAGTTCTGAAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT
VEGFA target site 3
GGTGAGTGAGTGTGTGCGTG TGG
Sequences used for gRNA synthesis are color coded to match Figure 2A.

7. Table S2. Delivery conditions in variety of cell lines
Plasmid
mRNA
Protein
Lipid
Electro
HEK293FT
3000
*-1150v/20ms/2pulses
MessengerMAX
RNAiMAX
U2OS
v/30ms/2pulses
v/20ms/2pulses
*-1400v/15ms/4pulses
Mouse ESCs
v/10ms/3pulses
Human ESCs (H9) -
17-850v/30ms/2pulses
9-1400v/30ms/1pulse
Human iPSCs
v/20ms/2pulse
N2A
v/30ms/2pulses
Jurkat
5-1700v/20ms/1pulse
K562
v/10ms/3pulses)
A549
*-1200v/20ms/4pulses
Human Keratinocytes (NHEK)
Human Cord Blood Cells CD34+
n/a
v/20ms/2pulses
For lipid mediated transfection or electroporation by Neon instrument, we tested Lipofectamine 3000, RNAiMAX or MessengerMAX in each listed cell line with each cas9 version (plasmid, mRNA or protein). For lipids, we listed the reagent that resulted in best cleavage efficiency reported in Table 1. In the electroporation column, the Neon optimization program number (Figure 3As) that performed best is listed followed by the specific conditions. Those conditions listed with an * resulted from further optimization. ‘n/a’ was not tested and ‘-’ means no successful conditions were found.

8. Table S3. Distribution of mutations induced by Cas9 and HPRT-T1 gRNA complexes in different cell lines
(A)
% wt
% base sub
%del
%ins
Total Sequenced
Jurkat T 5 4 16 75 81
Human iPSC 12 52 32 85
After editing with Cas9 RNP in Jurkat T or human iPSCs, the cells were lysed and the HPRT locus was PCR amplified Topo cloned. 96 clones were CE sequenced and aligned around the expected cleavage site (A). %base substitution is the clones that were the same length as wild type with mismatches at the cleavage site. %del is the clones whose overall length was shorter than wildtype. %ins is the clones whose overall length was longer than wild type. We also observed that some repairs were a result of partial deletion and insertion. Sequence alignments of the indels at the HPRT site for Jurkat T (B) and human iPSCs (C). The first sequence in alignment is wildtype HPRT, large deletions have the number of additional deleted bases annotated. Red bar over the GGG marks the PAM. Red arrows indicate location of expected cleavage.

9. (B) (C) Table S3. WT HPRT WT HPRT (-42) (-10) (-23) (-256) (-218)
(-81)
(-205)
(-194)
(-213)
(-201)
(-20)
(-18)
(-9)