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.

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.

(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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 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.

(A) 24 optimized protocol (C) Flow cytometry Figure S4 (A) 24 optimized protocol (C) Flow cytometry Unstained Control Tra -1-60- AlexaFluor®488 Cas9 mRNA Cas9 Protein mRNA Cas9 (B) Transfection via Electroporation Neg DNA mRNA 1 2 3 ug Cas9 SSEA4-AlexaFluor®647 % Cleavage: 0 20 31 62 80 89 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.

Figure S5. Time course of Cas9 RNP turnover and cleavage analysis in Mouse ESCs NC 1 2 4 6 8 10 12 h % Cleavage 0 7 8 13 21 37 39 43 (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.

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.

Table S2. Delivery conditions in variety of cell lines Plasmid mRNA Protein Lipid Electro HEK293FT 3000 *-1150v/20ms/2pulses MessengerMAX RNAiMAX U2OS 19-1050v/30ms/2pulses 16-1400v/20ms/2pulses *-1400v/15ms/4pulses Mouse ESCs 24-1600v/10ms/3pulses Human ESCs (H9) - 17-850v/30ms/2pulses 9-1400v/30ms/1pulse Human iPSCs 13-1100v/20ms/2pulse N2A 20-1150v/30ms/2pulses Jurkat 5-1700v/20ms/1pulse K562 24-1600v/10ms/3pulses) A549 *-1200v/20ms/4pulses Human Keratinocytes (NHEK) Human Cord Blood Cells CD34+ n/a 13-1100v/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.

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.

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