Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: Pathogenic DBD mutants introduced into the endogenous BRCA2 gene cause cytosolic mis-localization and disable DNA repair by HDR. ( A ) Subcellular fractionation of BRCA2 mutant cell lines, HeLa-W2626C and HeLa-D2723H. Two independent clones for each mutation were used. Nuclear and cytoplasmic fractions of BRCA2 and RAD51 were analysed by western blotting. MEK2 is a cytoplasmic marker. ( B ) mClover Lamin A HDR assay. Cells transfected with Lamin A-targeting sgRNA and mClover Lamin A donor constructs as shown in the schematic were analysed for mClover Lamin A-positive cells. Mean of HDR positive cells (%) ± s.e. from two repeats is shown. More than 100 cells per sample were analysed in each repeat. Statistical significance was tested by one-way ANOVA test, followed by Bonferroni's multiple comparison test.

Article Snippet: Cells grown on coverslips in six-well plates were transfected with sgRNA plasmid targeting Lamin A (pUC CBA-SpCas9.EF1a-BFP.sgLMNA, Addgene Plasmid #98971) and donor plasmid (pCAGGS Donor mClover-LMNA, Addgene Plasmid #98970).

Techniques: Fractionation, Mutagenesis, Clone Assay, Western Blot, Marker, Transfection, Construct, Comparison

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 crRNA plasmids generated in this study, we took the template from pSLQ5429_pUC_hU6-crScaffold_EF1α-BFPv3 (Addgene #155306) and cloned in individual crRNAs using restriction-ligation method.

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 crRNA plasmids generated in this study, we took the template from pSLQ5429_pUC_hU6-crScaffold_EF1α-BFPv3 (Addgene #155306) and cloned in individual crRNAs using restriction-ligation method.

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 crRNA plasmids generated in this study, we took the template from pSLQ5429_pUC_hU6-crScaffold_EF1α-BFPv3 (Addgene #155306) and cloned in individual crRNAs using restriction-ligation method.

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 crRNA plasmids generated in this study, we took the template from pSLQ5429_pUC_hU6-crScaffold_EF1α-BFPv3 (Addgene #155306) and cloned in individual crRNAs using restriction-ligation method.

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 crRNA plasmids generated in this study, we took the template from pSLQ5429_pUC_hU6-crScaffold_EF1α-BFPv3 (Addgene #155306) and cloned in individual crRNAs using restriction-ligation method.

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

Journal: bioRxiv

Article Title: A simple MiMIC based approach for tagging endogenous genes to visualise live transcription in vivo

doi: 10.1101/2024.08.29.610339

Figure Lengend Snippet: (Ai) Schematic showing the pxb gene locus with the 24xMS2V6 insertion. Males with the 24xMS2V6 loops inserted into pxb are crossed to females expressing HisRFP and 2xMCP2xmNeonGreen under the control of the nos promoter. Embryos have one of the two pxb alleles visible as a mNeonGreen puncta within the HisRFP nucleus, the imaging region is shown in yellow. (Aii) Top: Still images from a timelapse movie of pxb- 24xMS2V6 transcription sites in two anterior stripes in the nc14 embryo. Scale bar is 20µm. Bottom: Higher magnification image from nuclei with the stripe. Nuclei are marked in magenta and pxb- MS2 TSs are in green. Scale bar is 5µm. (Bi) As in (Ai) but with 24xPP7 loops inserted into pxb and females expressing HisBFP and 2xPCP2xmCherry. (Bii) Still from a movie of an embryo expressing HisBFP and 2xPCP2xmCherry marking the nuclei in blue and the pxb- PP7 TSs in magenta. (Ci) Schematic showing pxb labelled to visualise each allele with a different loop type (MS2 or PP7). (Cii) Dual imaging of these embryos detects transcription of both alleles. Pxb- PP7 TSs are in magenta, pxb- MS2 TSs are in green, nuclei are blue in the merge. See also Movies 1-3.

Article Snippet: Vectors pCR4-24xMS2SL-stable (Addgene #31865), pBlueScript-24xPP7 ( ) and pET264-pUC 24xMS2V6 Loxp KANr Loxp (Addgene #104393) were digested using BamHI and BglII to obtain 24xMS2-SL, 24xPP7 and 24xMS2V6 respectively.

Techniques: Expressing, Control, Imaging