methylation defective strain  (ATCC)

Journal: ACS Synthetic Biology

Article Title: Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting

doi: 10.1021/acssynbio.4c00271

Figure Lengend Snippet: Negative-feedback transcriptional regulation of both Cas13d protein and crRNA improves dose–response characteristics. (A) Diagram of single-vector transfection to test Cas13d’s Dox dose-dependent on-target activity. The plasmid encoding the BFP reporter and the BFP-targeting crRNA or nontargeting crRNA was transfected into HEK 293 cells with mNF-Cas13d stably LP-integrated. Cells were Dox-induced and incubated for 72 h before flow cytometry measurements. (B) Dox dose–responses of mean mCherry fluorescence intensity in HEK 293 cells with stably LP-integrated mNF-Cas13d gene circuit 72 h post-transfection. Unpaired two-tailed t test, n = 3, ** P < 0.01, *** P < 0.001. (C) Dox dose-dependent reduction of transiently transfected BFP reporter by Cas13d targeting. All BFP expression levels were normalized to uninduced cells with nontargeting guide, n = 3. (D) Diagram of single-vector transfection to test Cas13d’s Dox dose-dependent, on-target activity with regulated crRNA expression. BFP-targeting crRNA was driven by a modified human U6 (hU6) promoter containing two Tetracycline Operator (TetO2) sites flanking the TATA-box. This hU6-2O promoter can then be repressed by hTetR expressed from the mNF-Cas13d gene circuit. (E) Comparison of mCherry dose–responses from the mNF-Cas13d gene circuit 72 h post-transfection with all three transfection constructs. Unpaired two-tailed t test, n = 3, ** P < 0.01, *** P < 0.001. (F) Comparison of Dox dose-dependent BFP reduction by Cas13d, 72 h post-transfection with all three transfection constructs. All BFP expression levels were normalized to uninduced samples with nontargeting guide, n = 3. (G) Diagram of all-in-one constructs to test Cas13d’s on-target activity on stably genome-integrated targets. The GFP reporter-targeting crRNA is expressed from the TetR-regulated hU6-2O promoter, while the nontargeting guide is freely expressed from the normal hU6 promoter. The whole construct was FLP-RMCE integrated using the same HEK 293 LP parental cells. (H) Dose–responses of mean mCherry fluorescence intensity for stably integrated all-in-one constructs in engineered HEK 293 cells 72 h postinduction. Unpaired two-tailed t test, n = 3, ** P < 0.01, *** P < 0.001. (I) Dose-dependent reduction of stably integrated GFP reporter expression by Cas13d. All GFP expression levels were normalized to uninduced samples with nontargeting guide, n = 3.

Article Snippet: For constructing the RMCE vectors, we first built the pUt-mNF-Cas13d gene circuit plasmid by replacing the GFP reporter with the mCherry-P2A-RfxCas13d target cascade, obtained from pSLQ5428_pHR_EF1a-mCherry-P2A-Rfx_Cas13d-2xNLS-3xFLAG (Addgene #155305), in the previously cloned pUt-mNF-GFP RMCE plasmid (Addgene #199220).

Techniques: Plasmid Preparation, Transfection, Activity Assay, Stable Transfection, Incubation, Flow Cytometry, Fluorescence, Two Tailed Test, Expressing, Modification, Comparison, Construct

Journal: ACS Synthetic Biology

Article Title: Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting

doi: 10.1021/acssynbio.4c00271

Figure Lengend Snippet: Collateral activity due to Cas13d hyperactivation depends on Cas13d and crRNA expression levels. (A) Diagram of experimental setup to assess collateral activity from hyperactivated Cas13d with different guide control scenarios. The SV40 promoter-driven GFP target was cotransfected with either TetR-regulated GFP-targeting crRNA, constitutively expressed GFP-targeting crRNA or nontargeting crRNA. All three crRNA plasmids contain the same BFP gene driven by the EF1α promoter. Cells were transfected under 72 h induction before flow cytometry measurements. (B) Comparison of mCherry dose–responses from the mNF-Cas13d gene circuit 72 h post-transfection for all three cotransfection scenarios. Unpaired two-tailed t test, n = 3, ** P < 0.01. (C) Comparison of relative GFP levels indicating dose–responses of on-target activity from the mNF-Cas13d gene circuit 72 h post-transfection with all three cotransfection scenarios. All GFP expression levels were normalized to the uninduced sample with nontargeting guide, n = 3. (D) Comparison of relative BFP levels indicating dose–responses of off-target activity from the mNF-Cas13d gene circuit 72 h post-transfection with all three cotransfection setups. All the BFP expression levels were normalized to the uninduced sample with nontargeting guide, n = 3. (E) Diagram of all-in-one constructs to test Cas13d’s on-target and off-target activities on stably genome-integrated targets. GFP reporter-targeting crRNA is expressed from the hTetR-regulated hU6-2O promoter, while the nontargeting guide is constitutively expressed from the normal hU6 promoter. The whole construct is FLP-RMCE-integrated using the same HEK 293 LP parental cells. (F) Dose–responses of mCherry reporter indicating Cas13d expression levels for stably integrated all-in-one constructs in HEK 293 cells 72 h postinduction. Unpaired two-tailed t test, n = 3, * P < 0.05, ** P < 0.01. (G) Dose–responses of relative GFP reporter levels indicating on-target activity for stably integrated all-in-one constructs in HEK 293 cells 72 h postinduction. All GFP expression levels were normalized to uninduced samples with nontargeting guide, n = 3. (H) Dose–responses of relative BFP reporter levels indicating off-target activity for stably integrated all-in-one constructs in HEK 293 cells 72 h postinduction. All BFP expression levels were normalized to uninduced samples with nontargeting guide, n = 3.

Article Snippet: For constructing the RMCE vectors, we first built the pUt-mNF-Cas13d gene circuit plasmid by replacing the GFP reporter with the mCherry-P2A-RfxCas13d target cascade, obtained from pSLQ5428_pHR_EF1a-mCherry-P2A-Rfx_Cas13d-2xNLS-3xFLAG (Addgene #155305), in the previously cloned pUt-mNF-GFP RMCE plasmid (Addgene #199220).

Techniques: Activity Assay, Expressing, Control, Transfection, Flow Cytometry, Comparison, Cotransfection, Two Tailed Test, Construct, Stable Transfection

Journal: ACS Synthetic Biology

Article Title: Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting

doi: 10.1021/acssynbio.4c00271

Figure Lengend Snippet: Multilevel negative autoregulation of Cas13d and crRNA in MONARCH reduces the basal target downregulation. (A) Design and rationale for multilevel negative autoregulation of both Cas13d and crRNA expression, to avoid their unwanted overexpression, geberating too many activated Cas13d:crRNA complexes with excessive basal effect and collateral activity. Incorporating the crRNA into the same transcript with Cas13d not only brings both under the same tight transcriptional regulation, but also adds RNA-level regulation via crRNA processing by Cas13d. This may reduce the basal effect as well as the collateral activity. (B) Representative dose–responses of fluorescence intensity histograms from the stably integrated MONARCH 1.0 circuit measured at 0, 0.1, 0.2, 0.5, 1, 10 ng/mL Dox levels, respectively. (C) Dose–responses of mean mCherry fluorescence intensity for the MONARCH 1.0 circuit stably integrated into HEK 293 LP cells ( n = 3). (D) Dose–responses of the coefficient of variation (CV) of mCherry reporter expression for MONARCH 1.0 stably integrated into HEK 293 LP cells ( n = 3). (E) Diagram for testing on-target activity of Cas13d expressed from MONARCH 1.0 on stably genome-integrated targets. The whole construct is FLP-RMCE-integrated using the same HEK 293 LP parental cells. Basal level is determined with integration of only the SV40 promoter-driven GFP target. (F) Dose–responses of mean fluorescence intensity of mCherry reporter for stably integrated MONARCH 1.0_SV40-GFP construct in HEK 293 cells ( n = 3). (G) Dose–responses of coefficient of variation (CV) of mCherry reporter for stably integrated MONARCH 1.0_SV40-GFP in HEK 293 cells ( n = 3). (H) Dose–responses of relative GFP levels indicating on-target activity for the MONARCH 1.0_SV40-GFP construct stably LP-integrated into HEK 293 cells. All GFP expression levels were normalized to the basal sample, n = 3. (I) Dose–responses of coefficient of variation (CV) of GFP target expression for MONARCH 1.0_SV40-GFP stably LP-integrated into HEK 293 cells ( n = 3). (J) Diagram for testing on-target activity of MONARCH 1.0 on an endogenous target. GFP-targeting crRNA serves only as an RNA-level regulator in this scenario. Multiple crRNAs targeting BACH1 are cloned into a single plasmid and transfected together into the cells. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed after 144 h of induction and incubation post-transfection. (K) Dose-dependent reduction of endogenous BACH1 by Cas13d expressed from MONARCH 1.0 stably LP-integrated into HEK 293 cells. One-way ANOVA with Tukey’s multiple comparisons tests between each dose and native control, n = 3.

Article Snippet: For constructing the RMCE vectors, we first built the pUt-mNF-Cas13d gene circuit plasmid by replacing the GFP reporter with the mCherry-P2A-RfxCas13d target cascade, obtained from pSLQ5428_pHR_EF1a-mCherry-P2A-Rfx_Cas13d-2xNLS-3xFLAG (Addgene #155305), in the previously cloned pUt-mNF-GFP RMCE plasmid (Addgene #199220).

Techniques: Expressing, Over Expression, Activity Assay, Fluorescence, Stable Transfection, Construct, Clone Assay, Plasmid Preparation, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Incubation, Control

Journal: ACS Synthetic Biology

Article Title: Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting

doi: 10.1021/acssynbio.4c00271

Figure Lengend Snippet: Optimized multilevel negative autoregulation of Cas13d and crRNA improves the on-target potency and minimizes the basal effect. (A) Design and rationale for optimized multilevel negative-autoregulation for Cas13d and crRNA expression. Compared to MONARCH 1.0, a tertiary RNA structural motif “Triplex” is incorporated between the CDS (coding sequence) and crRNA to stabilize transcripts lacking poly(A) due to crRNA processing, enabling a moderately extended window of protein expression. (B) Representative dose–responses of fluorescence intensity histograms from the stably integrated MONARCH 2.0 circuit measured at 0, 0.1, 0.2, 0.5, 1, 10 ng/mL Dox levels, respectively. (C) Dose–responses of mean mCherry fluorescence intensity for the MONARCH 2.0 circuit stably LP-integrated into HEK 293 cells ( n = 3). (D) Dose–responses of coefficient of variation (CV) of the mCherry reporter for MONARCH 2.0 stably LP-integrated into HEK 293 cells ( n = 3). (E) Diagram for testing the on-target activity of Cas13d expressed from MONARCH 2.0 on genomically integrated targets. The whole construct is FLP-RMCE-integrated using the same HEK 293 LP parental cells. The basal level is determined through the integration of only SV40 promoter-driven GFP. (F) Dose–responses of mean mCherry fluorescence intensity for the MONARCH 2.0_SV40-GFP construct stably LP-integrated into HEK 293 cells ( n = 3). (G) Dose–responses of coefficient of variation (CV) of the mCherry reporter for MONARCH 2.0_SV40-GFP stably LP-integrated into HEK 293 cells ( n = 3). (H) Dose–responses of relative GFP levels indicating on-target activity for MONARCH 2.0_SV40-GFP stably LP-integrated into HEK 293 cells. All the GFP expression levels were normalized to the basal sample, n = 3. (I) Dose–responses of coefficient of variation (CV) of the GFP target for stably integrated MONARCH 2.0_SV40-GFP in HEK 293 cells ( n = 3). (J) Diagram for testing on-target activity of MONARCH 2.0 on an endogenous target. GFP-targeting crRNA serves only as an RNA-level regulator in this scenario. Multiple crRNAs targeting BACH1 are cloned into a single plasmid and transfected together into the cells. RT-qPCR was performed after 144 h of induction and incubation post-transfection. (K) Dose-dependent reduction of endogenous BACH1 by Cas13d expressed from MONARCH 2.0 stably LP-integrated into HEK 293 cells. One-way ANOVA with Tukey’s multiple comparisons test between each dose and native control, n = 3.

Article Snippet: For constructing the RMCE vectors, we first built the pUt-mNF-Cas13d gene circuit plasmid by replacing the GFP reporter with the mCherry-P2A-RfxCas13d target cascade, obtained from pSLQ5428_pHR_EF1a-mCherry-P2A-Rfx_Cas13d-2xNLS-3xFLAG (Addgene #155305), in the previously cloned pUt-mNF-GFP RMCE plasmid (Addgene #199220).

Techniques: Expressing, Sequencing, Fluorescence, Stable Transfection, Activity Assay, Construct, Clone Assay, Plasmid Preparation, Transfection, Quantitative RT-PCR, Incubation, Control

Journal: ACS Synthetic Biology

Article Title: Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting

doi: 10.1021/acssynbio.4c00271

Figure Lengend Snippet: MONARCH established in Vero E6 LP cells demonstrates antiviral capability and potential for improving on-target efficiency. (A) Diagram of experimental setup to repurpose MONARCH to target SARS-CoV-2 viral genome fragments in engineered Vero E6 cells. Both MONARCH systems are stably integrated into the AAVS1 ortholog in the Vero E6 genome using the same Landing-Pad and RMCE strategy. Successful by Cas13d targeting of SARS-CoV-2 viral genome fragments cloned into the 3′UTR of the GFP transcript will result in mRNA degradation, which can be measured by GFP fluorescence reduction. (B) Comparison of mCherry dose–responses from MONARCH 1.0 and 2.0 stably LP-integrated into Vero E6 cells 72 h post-transfection. (C) Comparison of coefficient of variation (CV) of mCherry dose–responses from MONARCH 1.0 and 2.0 stably integrated in Vero E6 cells 72 h post-transfection. (D) Comparison of relative GFP levels indicating dose–responses of on-target activity from MONARCH 1.0 and 2.0 stably LP-integrated into Vero E6 cells, 72 h post-transfection. All the GFP expression levels were normalized to native Vero E6 cells transfected with the target plasmid only. Unpaired two-tailed t test for each dosage comparison, n = 3, ** P < 0.01, *** P < 0.001. (E) Diagram of experimental setup to assess further improvement of MONARCH on-target activity by providing extra crRNA. Target donor plasmid is cotransfected with either SARS-CoV-2-targeting CRISPR array, nontargeting crRNA or blank control into the engineered Vero E6 cells. Engineered Vero E6 cells are preinduced with 10 ng/mL doxycycline 72 h before transfection. (F) Relative mCherry expression increases from 10 ng/mL doxycycline preinduced MONARCH 1.0 and 2.0 stably integrated into Vero E6 cells, 72 h post-transfection with extra crRNA plasmids. All mCherry expression levels were normalized to the Vero E6 cells transfected with a blank control plasmid. (G) Relative GFP level reductions 72 h post-transfection with extra crRNA plasmids. All GFP expression levels were normalized to the Vero E6 cells transfected with a blank control plasmid.

Article Snippet: For constructing the RMCE vectors, we first built the pUt-mNF-Cas13d gene circuit plasmid by replacing the GFP reporter with the mCherry-P2A-RfxCas13d target cascade, obtained from pSLQ5428_pHR_EF1a-mCherry-P2A-Rfx_Cas13d-2xNLS-3xFLAG (Addgene #155305), in the previously cloned pUt-mNF-GFP RMCE plasmid (Addgene #199220).

Techniques: Stable Transfection, Clone Assay, Fluorescence, Comparison, Transfection, Activity Assay, Expressing, Plasmid Preparation, Two Tailed Test, CRISPR, Control

Journal: bioRxiv

Article Title: Optimizing a CRISPR-Cas13d gene circuit for tunable target RNA downregulation with minimal collateral RNA cutting

doi: 10.1101/2024.05.11.593702

Figure Lengend Snippet: (A) Diagram of engineered HEK 293 cell with a single-copy landing pad located in the AAVS1 safe harbor site. FRT, FRT3: Flp-recombinase target sites; NeoR: Neomycin Resistance Gene; HSV-TK: Herpes Simplex Virus (HSV) thymidine kinase (TK); eGFP: enhanced Green Fluorescence Protein. (B) Left panel: Schematic diagram of repeatable AAVS1 site-specific integration of genetic payloads such as mNF-Cas13d gene circuit through Flp-recombinase-mediated cassette exchange (FLP-RMCE). Right panel: RMCE-based integration will result in a DNA construct switching between donor plasmid and target site. Successful integration of mNF-Cas13d gene circuit shifted the fluorescence signal from green (eGFP) to red (mCherry). HSV-TK in the donor plasmid backbone serves as a negative selection marker against random integration. (C) Diagram of synthetic mNF gene circuit for Dox-controlled tuning of co-expressed tetracycline repressor (TetR), mCherry reporter and Cas13d protein levels after site-specific integration. TetO: Tetracycline Operator; P2A: self-cleaving peptide. (D) Representative dose-responses of fluorescence intensity histograms from stably integrated mNF-Cas13d gene circuit measured at 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 100 ng/ml Dox levels, respectively. (E) Dose-responses of mean fluorescence intensity of mCherry reporter for stably integrated mNF-Cas13d gene circuit in HEK 293 cells (n=3). (F) Dose-responses of coefficient of variation (CV) of mCherry reporter for stably integrated mNF-Cas13d gene circuit in HEK 293 cells (n=3).

Article Snippet: For constructing the RMCE vectors, we first built pUt-mNF-Cas13d circuit plasmid by replacing the GFP reporter with mCherry-P2A-RfxCas13d target cascade, obtained from pSLQ5428_pHR_EF1a-mCherry-P2A-Rfx_Cas13d-2xNLS-3xFLAG (Addgene #155305), in the previous cloned pUt-mNF-GFP RMCE plasmid (Addgene #199220).

Techniques: Virus, Fluorescence, Construct, Plasmid Preparation, Selection, Marker, Stable Transfection

Journal: bioRxiv

Article Title: Optimizing a CRISPR-Cas13d gene circuit for tunable target RNA downregulation with minimal collateral RNA cutting

doi: 10.1101/2024.05.11.593702

Figure Lengend Snippet: (A) Diagram of single-vector transfection to test Cas13d dose-dependent on-target activity. Plasmid encoding BFP reporter and BFP targeting guide or non-targeting guide were transfected into mNF-Cas13d stably integrated HEK 293 cells with corresponding doxycycline (Dox) induction and incubated for 72 hours before flow cytometry. (B) Dose-responses of mean fluorescence intensity of mCherry reporter for stably integrated mNF-Cas13d gene circuit in HEK 293 cells 72 hours post-transfection. unpaired two-tailed t-test, n=3, **P<0.01, ***P<0.001. (C) Dose-dependent reduction of transiently transfected BFP reporter induced by Cas13d targeting. All the BFP expression levels were normalized to uninduced sample with non-targeting guide, n = 3. (D) Diagram of single-vector transfection to test Cas13d dose-dependent on-target activity with regulated crRNA expression. crRNA targeting BFP was expressed from a synthetically modified human U6 (hU6) promoter containing two Tetracycline Operator (TetO2) sites flanking the TATA-box. hU6-2O promoter can be then repressed by Tetracycline Repressor (TetR) expressed from mNF-Cas13d gene circuit. (E) Comparison of mCherry dose-responses from the mNF-Cas13d gene circuit 72 hours post-transfection with all three transfection constructs. unpaired two-tailed t-test, n=3, **P<0.01, ***P<0.001. (F) Comparison of dose-dependent BFP reduction induced by Cas13d targeting 72 hours post-transfection with all three transfection constructs. All the BFP expression levels were normalized to uninduced sample with non-targeting guide, n = 3. (G) Diagram of all-in-one constructs to test Cas13d on-target activity by stably expressing targets from the genome. crRNA targeting the GFP reporter is expressed from the TetR-regulated hU6 promoter while the non-targeting guide is freely expressed from the regular hU6 promoter. The whole construct is integrated via FLP-RMCE using the same HEK 293 Landing pad parental cells. (H) Dose-responses of mean fluorescence intensity of mCherry reporter for stably integrated all-in-one constructs in HEK 293 cells 72 hours post-induction. unpaired two-tailed t-test, n=3, **P<0.01, ***P<0.001. (I) Dose-dependent reduction of stably integrated GFP reporter by Cas13d targeting. All GFP expression levels were normalized to uninduced sample with non-targeting guide, n = 3.

Article Snippet: For constructing the RMCE vectors, we first built pUt-mNF-Cas13d circuit plasmid by replacing the GFP reporter with mCherry-P2A-RfxCas13d target cascade, obtained from pSLQ5428_pHR_EF1a-mCherry-P2A-Rfx_Cas13d-2xNLS-3xFLAG (Addgene #155305), in the previous cloned pUt-mNF-GFP RMCE plasmid (Addgene #199220).

Techniques: Plasmid Preparation, Transfection, Activity Assay, Stable Transfection, Incubation, Flow Cytometry, Fluorescence, Two Tailed Test, Expressing, Modification, Comparison, Construct

Journal: bioRxiv

Article Title: Optimizing a CRISPR-Cas13d gene circuit for tunable target RNA downregulation with minimal collateral RNA cutting

doi: 10.1101/2024.05.11.593702

Figure Lengend Snippet: (A) Diagram of experimental setup to assess collateral damage from activated Cas13d with different guide expression regulation. Target GFP expressed from SV40 promoter was co-transfected with either TetR-regulated GFP-targeting crRNA, constitutively expressing GFP-targeting crRNA or non-targeting crRNA. All three crRNA plasmids contain the same BFP gene expressed from EF1α promoter. Cells were transfected while induced 72 hours before flow cytometry. (B) Comparison of mCherry dose-responses from the mNF-Cas13d gene circuit 72 hours post-transfection with all three co-transfection setups. unpaired two-tailed t-test, n=3, **P<0.01. (C) Comparison of relative GFP levels indicating on-target activity dose-responses from the mNF-Cas13d gene circuit 72 hours post-transfection with all three co-transfection setups. All the GFP expression levels were normalized to uninduced sample with non-targeting guide, n=3. (D) Comparison of relative BFP levels indicating off-target activity dose-responses from the mNF-Cas13d gene circuit 72 hours post-transfection with all three co-transfection setups. All the BFP expression levels were normalized to uninduced sample with non-targeting guide, n=3. (E) Diagram of all-in-one constructs to test Cas13d on-target activity and off-target activity by stably expressing targets from the genome. crRNA-targeting the GFP reporter is expressed from the TetR-regulated hU6 promoter while the non-targeting guide is freely expressed from normal hU6 promoter. The whole construct is integrated via FLP-RMCE using the same HEK 293 Landing pad parentals. (F) Dose-responses of mCherry reporter indicating Cas13d expression levels for stably integrated all-in-one constructs in HEK 293 cells 72 hours post-induction. unpaired two-tailed t-test, n=3, *P<0.05, **P<0.01. (G) Dose-responses of relative GFP reporter levels indicating on-target activity for stably integrated all-in-one constructs in HEK 293 cells 72 hours post-induction. All the GFP expression levels were normalized to uninduced sample with non-targeting guide, n=3. (H) Dose-responses of relative BFP reporter levels indicating off-target activity for stably integrated all-in-one constructs in HEK 293 cells 72 hours post-induction. All the BFP expression levels were normalized to uninduced sample with non-targeting guide, n=3.

Article Snippet: For constructing the RMCE vectors, we first built pUt-mNF-Cas13d circuit plasmid by replacing the GFP reporter with mCherry-P2A-RfxCas13d target cascade, obtained from pSLQ5428_pHR_EF1a-mCherry-P2A-Rfx_Cas13d-2xNLS-3xFLAG (Addgene #155305), in the previous cloned pUt-mNF-GFP RMCE plasmid (Addgene #199220).

Techniques: Expressing, Transfection, Flow Cytometry, Comparison, Cotransfection, Two Tailed Test, Activity Assay, Construct, Stable Transfection

Journal: bioRxiv

Article Title: Optimizing a CRISPR-Cas13d gene circuit for tunable target RNA downregulation with minimal collateral RNA cutting

doi: 10.1101/2024.05.11.593702

Figure Lengend Snippet: (A) Design and rationale for multi-level negative-autoregulation for Cas13d and crRNA expression. Overexpression of both Cas13d and crRNA likely produce many activated Cas13d:crRNA complexes causing strong basal target reduction and collateral damage. Incorporating the crRNA into the same transcript with Cas13d not only brings both under the same tight transcriptional regulation but also adds a layer of RNA-level regulation via crRNA processing by Cas13d itself. This may greatly reduce the basal target reduction as well as the collateral damage. (B) Representative dose-responses of fluorescence intensity histograms from stably integrated MONARCH 1.0 circuit measured at 0, 0.1, 0.2, 0.5, 1, 10 ng/ml Dox levels, respectively. (C) Dose-responses of mean fluorescence intensity of mCherry reporter for stably integrated MONARCH 1.0 circuit in HEK 293 cells (n=3). (D) Dose-responses of coefficient of variation (CV) of mCherry reporter for stably integrated MONARCH 1.0 in HEK 293 cells (n=3). (E) Diagram to test on-target activity of Cas13d expressed from MONARCH 1.0 by stably expressing targets from the genome. The whole construct is integrated via FLP-RMCE using the same HEK 293 Landing pad parentals. Basal level is determined with integration of only GFP target expressed from the SV40 promoter. (F) Dose-responses of mean fluorescence intensity of mCherry reporter for stably integrated MONARCH 1.0_SV40-GFP construct in HEK 293 cells (n=3). (G) Dose-responses of coefficient of variation (CV) of mCherry reporter for stably integrated MONARCH 1.0_SV40-GFP in HEK 293 cells (n=3). (H) Dose-responses of relative GFP levels indicating on-target activity for stably integrated MONARCH 1.0_SV40-GFP construct in HEK 293 cells. All the GFP expression levels were normalized to the basal sample, n=3. (I) Dose-responses of coefficient of variation (CV) of target GFP reporter for stably integrated MONARCH 1.0_SV40-GFP in HEK 293 cells (n=3). (J) Diagram to test on-target activity of MONARCH 1.0 on the endogenous target other than the carrying crRNA. crRNA-targeting GFP serves only as a RNA-level regulator in this scenario. Multiple crRNAs targeting BACH1 are cloned into a single plasmid and transfected together. RT-qPCR was performed after 144 hours induction and incubation post-transfection. (K) Dose-dependent reduction of endogenous BACH1 induced by Cas13d targeting for stably integrated MONARCH 1.0 in HEK 293 cells. One-way ANOVA with Tukey’s multiple comparisons tests between each dose and native control, n=3.

Article Snippet: For constructing the RMCE vectors, we first built pUt-mNF-Cas13d circuit plasmid by replacing the GFP reporter with mCherry-P2A-RfxCas13d target cascade, obtained from pSLQ5428_pHR_EF1a-mCherry-P2A-Rfx_Cas13d-2xNLS-3xFLAG (Addgene #155305), in the previous cloned pUt-mNF-GFP RMCE plasmid (Addgene #199220).

Techniques: Expressing, Over Expression, Fluorescence, Stable Transfection, Activity Assay, Construct, Clone Assay, Plasmid Preparation, Transfection, Quantitative RT-PCR, Incubation, Control

Journal: bioRxiv

Article Title: Optimizing a CRISPR-Cas13d gene circuit for tunable target RNA downregulation with minimal collateral RNA cutting

doi: 10.1101/2024.05.11.593702

Figure Lengend Snippet: (A) Design and rationale for optimized multi-level negative-autoregulation for Cas13d and crRNA expression. Compared to the MONARCH 1.0, a tertiary RNA structural motif “Triplex” is incorporated between CDS and crRNA in order to stabilize transcripts lacking poly(A) due to crRNA processing, enabling moderately extended window of protein expression. (B) Representative dose-responses of fluorescence intensity histograms from stably integrated MONARCH 2.0 circuit measured at 0, 0.1, 0.2, 0.5, 1, 10 ng/ml Dox levels, respectively. (C) Dose-responses of mean fluorescence intensity of mCherry reporter for stably integrated MONARCH 2.0 circuit in HEK 293 cells (n=3). (D) Dose-responses of coefficient of variation (CV) of mCherry reporter for stably integrated MONARCH 2.0 in HEK 293 cells (n=3). (E) Diagram to test on-target activity of Cas13d expressed from MONARCH 2.0 by stably expressing targets from the genome. The whole construct is integrated via FLP-RMCE using the same HEK 293 Landing pad parentals. Basal level is determined with integration of only GFP target expressed from the SV40 promoter. (F) Dose-responses of mean fluorescence intensity of mCherry reporter for stably integrated MONARCH 2.0_SV40-GFP construct in HEK 293 cells (n=3). (G) Dose-responses of coefficient of variation (CV) of mCherry reporter for stably integrated MONARCH 2.0_SV40-GFP in HEK 293 cells (n=3). (H) Dose-responses of relative GFP levels indicating on-target activity for stably integrated MONARCH 2.0_SV40-GFP construct in HEK 293 cells. All the GFP expression levels were normalized to the basal sample, n=3. (I) Dose-responses of coefficient of variation (CV) of target GFP reporter for stably integrated MONARCH 2.0_SV40-GFP in HEK 293 cells (n=3). (J) Diagram to test on-target activity of MONARCH 2.0 on the endogenous target other than the carrying crRNA. crRNA-targeting GFP serves only as a RNA-level regulator in this scenario. Multiple crRNAs targeting BACH1 are cloned into a single plasmid and transfected together. RT-qPCR was performed after 144 hours induction and incubation post-transfection. (K) Dose-dependent reduction of endogenous BACH1 induced by Cas13d targeting for stably integrated MONARCH 2.0 in HEK 293 cells. One-way ANOVA with Tukey’s multiple comparisons tests between each dose and native control, n=3.

Article Snippet: For constructing the RMCE vectors, we first built pUt-mNF-Cas13d circuit plasmid by replacing the GFP reporter with mCherry-P2A-RfxCas13d target cascade, obtained from pSLQ5428_pHR_EF1a-mCherry-P2A-Rfx_Cas13d-2xNLS-3xFLAG (Addgene #155305), in the previous cloned pUt-mNF-GFP RMCE plasmid (Addgene #199220).

Techniques: Expressing, Fluorescence, Stable Transfection, Activity Assay, Construct, Clone Assay, Plasmid Preparation, Transfection, Quantitative RT-PCR, Incubation, Control

Journal: bioRxiv

Article Title: Optimizing a CRISPR-Cas13d gene circuit for tunable target RNA downregulation with minimal collateral RNA cutting

doi: 10.1101/2024.05.11.593702

Figure Lengend Snippet: (A) Diagram of experimental setup to repurpose the MONARCH system to target SARS-CoV-2 genome fragments in engineered Vero cells. Both MONARCH systems were stably integrated into the AAVS1 ortholog in Vero E6 genome using the same Landing-Pad and RMCE strategy. SARS-CoV-2 genome fragments are cloned into the 3’UTR of the GFP transcript, and successful targeting on them by Cas13d will result in mRNA degradation, which can be measured by GFP fluorescence reduction. (B) Comparison of mCherry dose-responses from MONARCH 1.0 and 2.0 stably integrated in Vero cell 72 hours post-transfection. (C) Comparison of coefficient of variation (CV) of mCherry dose-responses from MONARCH 1.0 and 2.0 stably integrated in Vero cell 72 hours post-transfection. (D) Comparison of relative GFP levels indicating on-target activity dose-responses from MONARCH 1.0 and 2.0 stably integrated in Vero cell 72 hours post-transfection. All the GFP expression levels were normalized to native Vero cells transfected with target plasmid only. Unpaired two-tailed t-test for each dosage comparison, n=3, **P<0.01, ***P<0.001. (E) Diagram of experimental setup to assess further improvement of MONARCH on-target activity by externally providing extra crRNA. Target donor plasmid is co-transfected with either SARS-CoV-2-targeting CRISPR array, non-targeting crRNA or blank control into the engineered Vero cells. Engineered Vero cells are pre-induced with 10 ng/ml doxycycline 72 hours before transfection. (F) Relative mCherry expression increases from 10 ng/ml doxycycline pre-induced MONARCH 1.0 and 2.0 stably integrated in Vero cell 72 hours post-transfection with extra crRNA plasmids. All the mCherry expression levels were normalized to the Vero cells transfected with Blank control plasmid. (G) Relative GFP levels reduction 72 hours post-transfection with extra crRNA plasmids. All the GFP expression levels were normalized to the Vero cells transfected with Blank control plasmid.

Article Snippet: For constructing the RMCE vectors, we first built pUt-mNF-Cas13d circuit plasmid by replacing the GFP reporter with mCherry-P2A-RfxCas13d target cascade, obtained from pSLQ5428_pHR_EF1a-mCherry-P2A-Rfx_Cas13d-2xNLS-3xFLAG (Addgene #155305), in the previous cloned pUt-mNF-GFP RMCE plasmid (Addgene #199220).

Techniques: Stable Transfection, Clone Assay, Fluorescence, Comparison, Transfection, Activity Assay, Expressing, Plasmid Preparation, Two Tailed Test, CRISPR, Control