Supplementary Materials for

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1 Supplementary Materials for CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation Xin Han, Zongbin Liu, Myeong chan Jo, Kai Zhang, Ying Li, Zihua Zeng, Nan Li, Youli Zu, Lidong Qin The PDF file includes: Published 14 August 2015, Sci. Adv. 1, e (2015) DOI: /sciadv Fig. S1. Performance of chip with different designs. Fig. S2. Cell stress simulation. Fig. S3. Flow velocity simulation. Fig. S4. Comparison of FuGENE HD transfection and delivery via chip. Fig. S5. Flow cytometric analysis of EGFP knockout cells. Fig. S6. Pten and 53BP1 knockout mediated by delivery via chip. Legends for movies S1 and S2 Other Supplementary Material for this manuscript includes the following: (available at Movie S1 (.avi format). Cells passing through the diamonded microconstrictions. Movie S2 (.avi format). Flow velocity simulation in the diamonded microconstriction chip.

2 Supplementary Materials Fig.S1 Fig.S1. Performance of chip with different designs. (A) Micro-constrictions formed by circular, elliptic, and diamonded microposts. (Scale bar: 20 µm.) (B) Delivery efficiency and (C) cell viability were calculated as a function of flow rate in three different micro-constrictions. FITC-labeled single strand DNA was delivered into HEK293T cells and cells were analyzed after culture for 16 h. Error bars indicate SEM. (n = 3). (D) Device image consisting of 4 parallel channel networks. Different dyes have been injected to aid visualization (Scale bar: 1 mm.) (E) The normalized cell count of HEK293T and SUM159 was calculated before and after perfusion through the chip.

3 Fig.S2 Fig.S2. Stress simulation of cell perfusion through diamonded micro-constrictions of 15µm depth and 4µm width. Graphical representation of stress gradient across cell membrane was shown at the indicated time points.

4 Fig.S3 Fig.S3. Flow velocity simulation of cell perfusion through diamonded micro-constrictions. Graphical representation of flow velocity gradient was shown at the indicated time points.

5 Fig.S4 Fig.S4. Comparison of FuGENE HD transfection and delivery via chip. (A-C) Plasmids encoding GFP was transfected with two different methods: FuGENE HD or delivery by chip. Cells are from HEK293T, MCF7, and SUM159 cell lines. Bright field (BF) and corresponding fluorescence (GFP) images were taken after 36 h treatment (Scale bar: 5 µm.) (D-E) Plasmids encoding GFP with Phosphoglycerate Kinase 1 (PGK) promoter were transfected with FuGENE HD or delivered by chip. Cells are SU-DHL-1 lymphoma cells and mouse embryonic stem cells AB 2.2 cells. Bright field (BF) or GFP fluorescent field images show cells morphology after 36 h treatment. (Scale bar: 5 µm.) (F) Cells from (E) delivered by chip were immunostained with Oct4 antibody. (Scale bar: 10 µm.)

6 Fig.S5 Fig.S5. Flow cytometric analysis of EGFP knockout cells. (A-B) Flow cytometry analysis was shown after EGFP stable expressing MDAMB231 and SU-DHL-1 lymphoma cells were delivered with plasmids encoding only Cas9 protein or both sgegfp and Cas9 protein for 7 days. MDAMB231 and SU-DHL-1 lymphoma cells serve as a negative control for EGFP fluorescence signal. The percentage of cells displaying EGFP fluorescence was shown in red. (C) The specific targeting region of sgegfp-1 in SU-DHL-1 lymphoma cell line was amplified by PCR and products were sent for TA cloning. "T1-T6" donates 6 different clones and PCR products were shown in a DNA gel. Arrow indicates the insertions in EGFP locus. "C" donates control with wild type EGFP locus.

7 Fig.S6 Fig.S6. Pten and 53BP1 knockout mediated by delivery via chip. (A) Illustration of sgpten targeting region at the first exon. The 20-bp target sequence is shown in red. The PAM sequence is shown in blue. (B) MCF7 cells delivered with plasmids encoding only Cas9 protein or both sgpten and Cas9 protein were cultured for 7 days and then immunostained by indicated antibodies. (Scale bar: 10 µm.) (C) Illustration of sg53bp1 targeting region at the 13th exon. The 20-bp target sequence is shown in red. The PAM sequence is shown in blue. (D) Hela cells delivered with plasmids encoding only Cas9 protein or both sg53bp1 and Cas9 protein were cultured for 7 days and analyzed by western blotting using indicated antibodies. Actin was used as loading control. The symbol * indicated long exposure.

8 Movie S1. Movie for cells passing through the diamonded micro-constrictions at fluid speed of 30 μl/min. Movie S2. Movie for flow velocity simulation in the diamonded micro-constriction chip.