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, 46 (5), e25

Multimode Drug Inducible CRISPR/Cas9 Devices for Transcriptional Activation and Genome Editing

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Multimode Drug Inducible CRISPR/Cas9 Devices for Transcriptional Activation and Genome Editing

Jia Lu et al. Nucleic Acids Res.

Abstract

Precise investigation and manipulation of dynamic biological processes often requires molecular modulation in a controlled inducible manner. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) has emerged as a versatile tool for targeted gene editing and transcriptional programming. Here, we designed and vigorously optimized a series of Hybrid drug Inducible CRISPR/Cas9 Technologies (HIT) for transcriptional activation by grafting a mutated human estrogen receptor (ERT2) to multiple CRISPR/Cas9 systems, which renders them 4-hydroxytamoxifen (4-OHT) inducible for the access of genome. Further, extra functionality of simultaneous genome editing was achieved with one device we named HIT2. Optimized terminal devices herein delivered advantageous performances in comparison with several existing designs. They exerted selective, titratable, rapid and reversible response to drug induction. In addition, these designs were successfully adapted to an orthogonal Cas9. HIT systems developed in this study can be applied for controlled modulation of potentially any genomic loci in multiple modes.

Figures

Figure 1.
Figure 1.
Design, optimization and cross-comparison of HIT transcription activation systems. (A–C) Cartoons illuminating the mechanisms of optimized 4-OHT induced transcription activation using a direct fusion HIT construct (A), the HIT-SAM system (B), and the HIT-SunTag system (C). (D–I) Cross-comparison among three optimized HIT transcription activation systems. Transcription activation induced by HIT constructs was examined in the luciferase reporter assay (D), by quantification of relative mRNA level of endogenous expression for Klf4 (E) and Oct4 (F), and by flow-cytometry analyses of CD43 protein level on the cell surface (G–I). Representative plots (G) and quantitative analyses of median CD43 fluorescent intensities (H) and overall CD43 fluorescent intensities (I) of CD43+GFP+ positive population were shown. GFP fluorescence indicates successful transfection. Cells transfected with the same amount of reporter construct or sgRNA only while keeping the total amount of transfection constant were used as negative controls (NC) in the luciferase reporter assay. Cells transfected with an ERT2 tagged GFP construct while keeping the total amount of transfection constant were used as negative control (NC) in qRT-PCR assays. Cells transfected with an unrelated sgRNA (ctl sgRNA) and GFP were used as negative control (NC) in flow-cytometry analyses. Data showed mean ± SD. n = 3 biological replicates. ns: non-significant;*P < 0.05; **P < 0.01; ***P < 0.001; two tailed t-tests. Three biological replicates means three independently transfected samples throughout this study. The readouts without drug induction were compared in t-tests against the negative controls (NCs) for background detection.
Figure 2.
Figure 2.
HIT2: one CRISPR/Cas9 device for simultaneous genome editing and transcriptional activation in a drug inducible manner. (A) Cartoon illuminating the mechanism of the optimized drug inducible HIT2 system for simultaneous genome editing and transcriptional activation. (B) Simultaneous editing and activation by HIT2 and Cas9–NLS–GCN4 were examined using flow-cytometry. The percentage of GFP positive cells indicated HDR efficiency, while CD43 protein level on the cell surface represented transcription activation. Representative plots (upper panels) and quantitative analyses (lower panels) were shown. Data showed mean ± SD. n = 3 biological replicates. ns: non-significant;*P < 0.05; *** P < 0.001; ****P < 0.0001; two-tailed t-tests.
Figure 3.
Figure 3.
Comparisons of HIT systems with existing drug inducible designs. (A–D) Drug inducible efficiency and background activity of transcription activation was examined head-to-head between HIT systems and existing designs using the luciferase reporter assay (A), in which the expression of luciferase was controlled by a sgRNA target sequence (gLuc sgRNA), and the CD43 activation assay (B-D).Representative plots (B), quantitative analyses of the percentage of CD43 positive cells (C), and median CD43 fluorescent intensities (D) were shown. (E) Drug inducible efficiency and background activity of genome editing was examined using the FCR assay in comparison with existing designs. Representative plots (top) and quantifications (right bottom) were shown. NC, cells transfected with an unrelated sgRNA; PC, cells transfected with Cas9-NLS and BFP sgRNA. Data showed mean ± SD. n = 3 biological replicates. ns: non-significant;*P < 0.05; **P < 0.01; ****P < 0.0001; two-tailed t-tests.
Figure 4.
Figure 4.
Selective and titratable drug induction of HIT systems. (A and B) Dose dependent transcription activation induced by HIT-SunTag (A) and HIT2 (B) with different concentration of β-estradiol or 4OHT was examined using the luciferase reporter assay. (C and D) Activation of endogenous gene CD43 was examined using flow cytometry. Quantitative analyses of the percentage of CD43 positive cells (C) and median CD43 fluorescent intensities (D) were shown. (E) Dose dependent genome editing activities of HIT2 were examined using the FCR assay upon treatment with different concentration of β-estradiol or 4-OHT. Data showed mean ± SD. n = 3 biological replicates. ns: non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; two-tailed t-tests. Fold of activation by 4-OHT over the same concentration of β-estradiol was displayed.
Figure 5.
Figure 5.
Rapid and reversible drug induction of HIT systems. (A and B) Luciferase signal was examined upon drug treatment for distinct lengths of time using (A) HIT-SunTag system and (B) HIT2 system. (C) Reversibility of HIT-SunTag was examined in a stable cell line expressing the luciferase reporter and HIT-SunTag constructs. Cells were either continuously treated with 250 nM of 4-OHT for 9 days (ON9) or without (OFF9), or treated for 3 days followed by being cultured without 4-OHT for 6 days (ON3OFF6) or treated for 3 days, then cultured without 4-OHT for 3 days followed by the treatment of 4-OHT again for 3 days (ON3 OFF3 ON3), respectively. Data showed mean ± SD. n = 3 biological replicates. ns: non-significant;*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; two-tailed t-tests. The signals activated by HIT systems were compared against the negative controls (NCs).
Figure 6.
Figure 6.
Adaptation of HIT designs to SaCas9. (A and B) Drug inducible gene activation by NLS-dSaCas9–GCN4 in conjunction with scFv-2ERT2-AD was examined in the luciferase reporter assay (A) and the endogenous CD43 activation assay (B). (C) Simultaneous genome editing and transcriptional activation by HIT2-SaCas9 were examined by the FCR activity and CD43 activation respectively. Cells transfected with the same amount of reporter construct while keeping the total amount of transfection constant were used as negative controls (NC). ISO represents cells stained with antibody isotype control. Data showed mean ± SD. n = 3 biological replicates. ns: non-significant;*P < 0.05; **P < 0.01; ***P < 0.001; two-tailed t-tests.

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