Journal: ACS Central Science

Article Title: Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes

doi: 10.1021/acscentsci.0c00129

Figure Lengend Snippet: Demonstration of Cas9 degradation by dTAG-47. (A) Schematic showing the chemogenetic system to control Cas9 longevity using the small molecule, dTAG-47. Cas9 is fused with multiple (FKBP)12 F36V domains and investigated for dTAG-induced proteasomal degradation. (B) Dose-dependent and dTAG-47-induced Cas9 degradation in HEK293T cells transiently transfected with NL-FKBP-Cas9. (C) Upper panel: schematic of eGFP knockout assay to investigate Cas9 activity. Lower panel: eGFP disruption in U2OS.eGFP.PEST cells nucleofected with 10 pmol of ribonucleoprotein (RNP). The matched gRNA and the mismatched gRNA (1–3) were incubated with Cas9 or NL-FKBP-Cas9 to form the RNP. (D) dTAG-47 dose-dependent degradation of NL-FKBP-Cas9 in U2OS.eGFP.PEST cells measured by Cas9 activity in the eGFP disruption assay. (E) Top panel: schematic of knock-in of HiBiT ssODNs into GAPDH locus in HEK293T CRBN −/– and CRBN +/+ cell lines. GAPDH-HiBiT fusion protein will form a split NanoLuc protein upon complementation with LgBiT. Bottom panel: dTAG-47 dose-dependent (0, 1, 10, 100, 1000, 3000 nM) decrease in luminescence activity in CRBN +/+ cells but no change in the luminescence levels in the CRBN −/– cells, indicating that CRBN mediates Cas9 degradation. (F) Upper panel: schematic of eGFP knockout assay in Drosophila ’s S2 cells by measuring the indels using a T7E1 assay to investigate Cas9 activity. Lower panel: dose-dependent decrease in the indel formation due to degradation of NL-FKBP-Cas9 in S2 cells.

Article Snippet: For plasmid transfection, approximately 20 000 U2OS.eGFP-PEST cells were nucleofected with 300 ng of Cas9 expression plasmid and 30 ng of gRNA expression plasmid using an SE Cell line 4D-Nucleofector X kit (Lonza) following the manufacturer’s protocol.

Techniques: Transfection, Knock-Out, Activity Assay, Incubation, Knock-In

Journal: ACS Central Science

Article Title: Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes

doi: 10.1021/acscentsci.0c00129

Figure Lengend Snippet: Cas9 lifetimes impact DNA repair outcomes. (A) Deep-sequencing analysis of non-MH deletions and MH deletions, both raised from the NHEJ pathway. The mESC cell line with stable Reduced Library genomic integration was transfected with NL-FKBP-Cas9 plasmid. Then, 1 μM dTAG-47 was added at different time points after transfection (0–48 h) before genomic DNA was extracted at 120 h post-transfection. (B) ddPCR quantification of single-nucleotide exchange at the RBM20 locus in HEK293T cells following templated DNA repair. For this, 400 ng of NL-FKBP-Cas9 plasmid, 400 ng of RBM20 gRNA plasmid, and 40 pmol of ssODN were transfected to 0.2 × 10 6 HEK293T cells. dTAG-47 was introduced 0.5, 1, 2, 4, 8, 12, 24, and 48 h after transfection, and this was incubated until 72 h post-transfection. Cells were harvested at 72 h post-transfection, and percentages of HDR and NHEJ in the genomic DNA were analyzed by ddPCR analysis. At 72 h, no dTAG-47 addition occurred. (C) Luminescence-based quantification of HiBiT knock-in at the GAPDH locus in HEK293T cells following templated DNA repair. For this, 400 ng of NL-FKBP-Cas9 plasmid, 40 ng of GAPDH gRNA plasmid, and 40 pmol of ssODN were transfected to 0.2 × 10 6 HEK293T cells. dTAG-47 was introduced at 0, 0.5, 1, 2, 4, 8, 12, 24, and 48 h after transfection and was incubated until 72 h post-transfection. Cells were lysed at 72 h post-transfection and complemented with LgBiT protein to measure the luminescence. At 72 h, no dTAG-47 addition occurred.

Article Snippet: For plasmid transfection, approximately 20 000 U2OS.eGFP-PEST cells were nucleofected with 300 ng of Cas9 expression plasmid and 30 ng of gRNA expression plasmid using an SE Cell line 4D-Nucleofector X kit (Lonza) following the manufacturer’s protocol.

Techniques: Sequencing, Transfection, Plasmid Preparation, Incubation, Knock-In

Journal: Protein & Cell

Article Title: An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

doi: 10.1007/s13238-016-0360-8

Figure Lengend Snippet: DCas9-VPR can be used to activate single or multiple genes in 293FT cells . (A) NANOG gRNA targeting sites were located at -254 bp and -144 bp upstream of the NANOG transcription starting site (TSS); protospacer-adjacent motif (PAM) sequences in red; black boxes indicate exons. (B) DCas9-VPR and gNANOG plasmids were co-transfected into 293FT cells. DCas9-VPR and gTetO plasmids were used as control. Top panels, fluorescence images of transfected cells; gNANOG plasmid transfected cells showed strong GFP fluorescence. Bottom panel, flow cytometry analysis of GFP + cells in each group. (C) Q-PCR analysis of NANOG expression 2 days after transfection; the dCas9-VPR system showed nearly 150-fold up-regulation of NANOG mRNA. Relative gene expression values were normalized against GAPDH . Error bars represent SEM. ** P < 0.01, n = 3. (D) Activation of endogenous genes by dCas9-VPR. DCas9-VPR was co-transfected with gRNA pairs targeting HOXA10 , SNAIL1 , MESP1 , GATA5 or HOXA9 , respectively. Cells were harvested 2 days after transfection and subjected to Q-PCR analysis. All tested genes showed significant upregulation compared to the control group. All expression levels were normalized against GAPDH . Error bars represent SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 , n = 3. (E) Simultaneously activation of multiple endogenous genes in 293FT cells. DCas9-VPR was co-transfected with 2× gRNAs (gMESP1, gGATA5), 3× gRNAs (gHOXA10, gSNAIL1, gHOXA9) or 5× gRNAs (gHOXA10, gSNAIL1, gMESP1, gGATA5 and gHOXA9). Cells were harvested 2 days after transfection. Q-PCR analysis confirmed co-upregulation of multiple genes targeted by pooled gRNAs. All expression levels normalized against GAPDH . Error bars represent SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001 , n = 3

Article Snippet: The multiple NANOG gRNA expression plasmid was constructed by SynGene ( http://syngen.tech ) as depicted in Fig. A.

Techniques: Transfection, Fluorescence, Plasmid Preparation, Flow Cytometry, Expressing, Activation Assay

Journal: Protein & Cell

Article Title: An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

doi: 10.1007/s13238-016-0360-8

Figure Lengend Snippet: Upregulation of NANOG by dCas9-VPR promoted clonogenicity and the naïve state of pluripotency . (A) Q-PCR analysis of NANOG upregulation in iNANOG cells. IVPR clones 2, 6, and 8 were electroporated with gRNA expression plasmid targeting the NANOG promoter, as shown in Fig. A. GFP positive cells were purified by FACS and maintained as iNANOG cells. They were treated with or without Dox (1 μg/mL) for 2 days. NANOG expression level was normalized against GAPDH . Error bar represents SEM. (B) Q-PCR analysis of NANOG down-regulation in iNANOG cells. Dox was added for 2 days, then removed. Cells were harvested at different time points, as indicated. NANOG expression was normalized against GAPDH . Error bar represents SEM. (C) Western blot showing increased NANOG protein expression in iNANOG cells at different time points after Dox treatment. SE, short exposure; LE, long exposure; d, day; h, h. (D) Western blot showing NANOG protein expression decrease in iNANOG cells at different time points after Dox withdrawal. SE, short exposure; LE, long exposure; d, day; h, h. (E) Q-PCR analysis showing upregulation of pluripotency gene OCT4, PRDM14, GDF3, and LEFTYB, and down-regulation of differentiation gene AFP . Expression level all normalized against GAPDH . Error bar represents SEM. N/A, not applicable. ns. P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 , n = 3. (F) Q-PCR analysis showing downregulation of XIST after NANOG induction. Expression level normalized against GAPDH . Error bar represents SEM. ns. P > 0.05, * P < 0.05 , n = 3. (G) Flow cytometry analysis showing increased SSEA3 expression after NANOG induction. Data analyzed using the FlowJo software v7.6.1. (H) Clonogenicity assay of iNANOG cells. Alkaline phosphatase assay (dark blue) was used to visualize undifferentiated colonies. (I) Bar graph quantification of the clonogenicity assay. ns. P > 0.05, * P < 0.05 , n = 3. (J) Morphology of iNANOG cells cultured in the 2iL medium. NANOG overexpression (iNANOG + Dox) promoted long-term cell growth in the 2iL medium. Representative images of passages 1 and 8 (P1 and P8) are shown. Scale bar, 100 μm. (K) Morphology of primed state iNANOG cells (without Dox) and Dox induced iNANOG cells (passage 9, P9) in the 2iL medium

Article Snippet: The multiple NANOG gRNA expression plasmid was constructed by SynGene ( http://syngen.tech ) as depicted in Fig. A.

Techniques: Clone Assay, Expressing, Plasmid Preparation, Purification, Western Blot, Flow Cytometry, Software, ALP Assay, Cell Culture, Over Expression

Journal: Protein & Cell

Article Title: An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

doi: 10.1007/s13238-016-0360-8

Figure Lengend Snippet: Upregulation of NANOG by idCas9-VPR promoted hESC survival and ICM integration in mouse blastocysts in vitro . (A) Cartoon showing in vitro hESC-mouse blastocyst chimera formation assay. iNANOG cells were cultured in E8 or 2il/FK medium with or without Dox, then injected into E3.5 mouse blastocysts and cultured to E4.5 in KSOM: 2il/FK = 1:1 medium in vitro . (B) Morphology of iNANOG cells in culture and chimeric embryos. Top 2 rows, cells cultured in E8 or 2iL/FK on feeders; scale bar, 100 μm. Bottom 2 rows, E3.5 and E4.5 mouse blastocysts with iNANOG cells (GFP); scale bar, 100 μm. (C) Survival curve of hESC in mouse blastocysts over time. WTSG, wild type hESCs expressing NANOG gRNA. The P value was calculated using the Log-rank (Mantel-Cox) test. ns. P > 0.05, * P < 0.05, *** P < 0.001. Detailed information is provided in Fig. S4B. (D) Confocal images of E4.5 chimeric embryos. β-Catenin, yellow; CDX2, red; DNA, blue; iNANOG cells, green. The ICM region is highlighted by a dashed circle. Scale bar, 20 μm. (E) Selected frame from time-lapse movie of iNANOG-mouse blastocyst chimera. Arrows indicating iNANOG cells moved with mouse inner cell mass cells during blastocyst hatching. Scale bar, 100 μm. (F) The proportion of blastocysts with hESC integration for E4.5 embryos. Blastocysts with GFP cells in both ICM and TE were counted as ICM. The percentage of embryos with ICM/TE/None integration was labeled in the colored bar. (G) Dot graph showing the number of iNANOG cells in the ICM region of E4.5 embryos. Cells, culture condition before injection, and number of embryos were as listed. Error bars represent SEM. ns. P > 0.05, ** P < 0.01

Article Snippet: The multiple NANOG gRNA expression plasmid was constructed by SynGene ( http://syngen.tech ) as depicted in Fig. A.

Techniques: In Vitro, Tube Formation Assay, Cell Culture, Injection, Expressing, Labeling