guide rna lentiviral expression vector  (Addgene inc)

Journal: Nature Communications

Article Title: Precision multiplexed base editing in human cells using Cas12a-derived base editors

doi: 10.1038/s41467-025-59653-x

Figure Lengend Snippet: A The ability of Cas12a to process gRNA arrays without accessory factors allows for the efficient delivery of many gRNAs into a single cell. Truncated gRNAs can be used to prevent deamination of neighboring bases. Created in BioRender. Schweitzer, A. (2025) https://BioRender.com/z2a6bsw . B Comparison of two published dLbCas12a-derived CBE and ( C ), one published dLbCas12a-derived ABE system for MBE in HEK293 cells. Heatmaps show normalized mean %T/%G values from three independent replicates ( n = 3). Normalization was performed by subtracting the mean %T/%G values of the nt-ctrl condition from the mean %T/%G values of the experimental condition. Only positions 7–12 for CBEs and positions 8–12 of ABEs are shown, as those correspond to the editing window of the used systems. sg: single guide, dg: double guide, tg: triple guide. Source data are provided as a Source Data file.

Article Snippet: For co-transfection experiments, HEK293 cells were transfected with 340 ng gRNA expression plasmid and 500 ng base editor plasmid (Addgene IDs 171697, 171698, 138504, 138506, 114081, and 114082, Supplementary Data ).

Techniques: Comparison, Derivative Assay

Journal: Nature Communications

Article Title: Precision multiplexed base editing in human cells using Cas12a-derived base editors

doi: 10.1038/s41467-025-59653-x

Figure Lengend Snippet: A Schematic of the architecture of the three gRNA expression plasmids used in this study. SV40: Simian Virus 40 termination signal. B Schematic of gRNA array architectures used in this study. The standard array consists of the DR and guide sequences and does not include any additional sequence elements. The SynSep array includes an AAAT sequence upstream of each DR sequence, and the VarSep arrays include variable 4 nt sequences upstream of each DR sequence (see Supplementary Data ). C Comparison of different array architectures for BEACON2-mediated multiplex editing of 14 RUNX1 target sites. D Editing frequencies of gRNAs across arrays shown in ( C ), normalized to their efficiency when expressed using the standard array architecture. E BEACON2-mediated multiplex editing of 3–16 target sites, across six genes located on five chromosomes. F , Comparison of a Pol-III promoter (hU6) and two Pol-II promoters (CMV and EFs1a) for the expression of the standard gRNA array shown in ( D ). All data are the mean ± SD of three independent replicates ( n = 3). C – F show normalized mean %T values at the highest edited C for each gRNA as determined based on the single gRNA condition. Normalization was performed by subtracting the mean %T of the non-targeting (nt-ctrl) condition from the mean %T of the experimental condition. Source data are provided as a Source Data file.

Article Snippet: For co-transfection experiments, HEK293 cells were transfected with 340 ng gRNA expression plasmid and 500 ng base editor plasmid (Addgene IDs 171697, 171698, 138504, 138506, 114081, and 114082, Supplementary Data ).

Techniques: Expressing, Virus, Sequencing, Comparison, Multiplex Assay

Journal: Nature Communications

Article Title: Precision multiplexed base editing in human cells using Cas12a-derived base editors

doi: 10.1038/s41467-025-59653-x

Figure Lengend Snippet: A Total number of mutations found in three edited clones and one clone transfected with a non-targeting (nt-ctrl) gRNA. B Total number of detected single-nucleotide variants (SNVs) by base-change for three edited clones and one clone transfected with an nt-ctrl gRNA. C Summary of allele frequencies observed at the 16 targeted sites after editing with the 16x gRNA array (Fig. ), showing combined outcomes based on the number of edited Cs. Data represents three cell clones isolated from the edited population. D Total number of edited target sites per clone. E Overlap of C > T/G > A SNVs detected in our analysis and predicted off-target sites by Cas-OFFinder.

Article Snippet: For co-transfection experiments, HEK293 cells were transfected with 340 ng gRNA expression plasmid and 500 ng base editor plasmid (Addgene IDs 171697, 171698, 138504, 138506, 114081, and 114082, Supplementary Data ).

Techniques: Clone Assay, Transfection, Isolation

Journal: Nature Communications

Article Title: Precision multiplexed base editing in human cells using Cas12a-derived base editors

doi: 10.1038/s41467-025-59653-x

Figure Lengend Snippet: A Editing outcomes mediated by BEACON1 and BEACON2 when used with a triple gRNA array targeting RUNX1 . B Editing outcomes mediated by BEACON1 and BEACON2 at 16 target sites in HeLa cells transfected with a CMV promoter-driven 16x gRNA array and the respective BE system. Editing outcomes in HEK293-B2 cells mediated by BEACON2 transfected with the same 16 × gRNA array as presented in Fig. . A , B show normalized mean %T values at the highest edited C for each gRNA as determined based on the single gRNA condition. Normalization was performed by subtracting the mean %T of the non-targeting (nt-ctrl) condition from the mean %T of the experimental condition. Source data are provided as a Source Data file.

Article Snippet: For co-transfection experiments, HEK293 cells were transfected with 340 ng gRNA expression plasmid and 500 ng base editor plasmid (Addgene IDs 171697, 171698, 138504, 138506, 114081, and 114082, Supplementary Data ).

Techniques: Transfection

Journal: Nature Communications

Article Title: hnRNPL phase separation activates PIK3CB transcription and promotes glycolysis in ovarian cancer

doi: 10.1038/s41467-025-60115-7

Figure Lengend Snippet: a Spearman’s correlation analysis between mRNA levels of hnRNPL and PIK3CB according to the GEO ovarian cancer cohorts. b Schematic diagram of the luciferase reporters carrying wild-type or mutated PIK3CB promoter. c Analysis of luciferase activities of the reporters carrying wild-type or mutated PIK3CB promoters normalized to renilla luciferase activity in ovarian cancer cells with knockdown of hnRNPL. d Effect of hnRNPL loss on RNA Pol II enrichment at the promoter of PIK3CB . e Effect of wild-type or mutated hnRNPL overexpression on the PIK3CB mRNA level. f ChIP assays were performed to evaluate the enrichment of wild-type or mutated hnRNPL at the PIK3CB promoter in OVCAR3 cells by using the antibody specific to hnRNPL. g Schematic diagram of PI3K-AKT regulated glycolytic signaling pathway. h Western blot of PIK3CB and PI3K-AKT signaling pathway-associated proteins in ovarian cancer cells upon overexpression of wild-type or mutated hnRNPL. i Glucose content and lactate production in the culture medium were monitored in ovarian cancer cells upon overexpression of wild-type or mutated hnRNPL. j ECAR levels were determined in wild-type ovarian cancer cells and ovarian cancer cells with overexpression of hnRNPL variants of WT, IDR1 delG , IDR2 delP by the Seahorse assays. Data are shown as means ± S.D. p value was calculated by one-way ANOVA test with multiple comparisons ( c, e, f, i, j ) and unpaired two-sided Student’s t -test ( d ). Data are representative of at least three independent experiments ( c –f, h–j ). Source data are provided as a Source Data file.

Article Snippet: The sequences of oligonucleotides were shown in Supplementary materials. hnRNPL-WT, hnRNPL-IDR1 delG , hnRNPL-IDR2 delP , and hnRNPL-IDR2 FUS expression plasmids were constructed via cloning the corresponding sequence into pCDH-GFP-puro-3×flag, pcDNA3.1-mCherry, and pcDNA3.1-3×flag vector, respectively. hnRNPL-RRM1-1del, hnRNPL-RRM1-2del, hnRNPL-RRM5del and PIK3CB expression plasmids were constructed via cloning corresponding sequence into pcDNA3.1-3×flag vector, respectively. pHR-mCh-Cry2WT was a gift from Clifford Brangwynne (101221, Addgene; http://n2t.net/addgene:101221 ; RRID: Addgene, 101221). pHR-hnRNPL-IDR2 FUS -mCh-Cry2WT, pHR-hnRNPL-WT-mCh-Cry2WT, pHR-hnRNPL-IDR1 delG -mCh-Cry2WT, pHR-hnRNPL-IDR2 delP -mCh-Cry2WT expression plasmids were constructed via cloning corresponding sequence into pHR-mCh-Cry2WT vector, respectively.

Techniques: Luciferase, Activity Assay, Knockdown, Over Expression, Western Blot

Journal: Nature Communications

Article Title: hnRNPL phase separation activates PIK3CB transcription and promotes glycolysis in ovarian cancer

doi: 10.1038/s41467-025-60115-7

Figure Lengend Snippet: a RNase-ChIP assays were performed to confirm that hnRNPL bound to the promoters of IRS1 , CDK6 , PIK3CB and PCK2 depending RNAs. b RIP-PCR detecting the interaction between hnRNPL and the promoter-associated RNA paPIK3CB by using the antibody specific to hnRNPL in wild-type ovarian cancer cells. c Schematic diagram of the RRM-deleted hnRNPL constructs (RRM1-1del, RRM1-2del and RRM5del). d, e The interaction of the RRM-deleted hnRNPL constructs with paPIK3CB was examined by RIP assays. f Colocalization between wild-type hnRNPL or RRM-mutants and paPIK3CB RNA was examined in ovarian cancer cells under confocal microscopy by RNA FISH and IF assays. Scale bar, 5 µm. g Schematic of products from hnRNPL eCLIP-qPCR across paPIK3CB RNA. h eCLIP-qPCR by using an antibody specific to hnRNPL was performed in OVCAR3 ovarian cancer cells. i Schematic of wild-type paPIK3CB RNA and its three deletion mutants. j, k RIP assays were performed by using an antibody specific to hnRNPL in OVCAR3 ovarian cancer cells with overexpression of wild-type paPIK3CB RNA and its three deletion mutants. hnRNPL immunoprecipitated was assessed by western blot ( j ), and interaction between hnRNPL and wild-type paPIK3CB RNA or its mutants was detected by RT-qPCR ( k ). l Relative RNA expression of PIK3CB was detected upon paPIK3CB was knocked down by ASOs. m ChIP asssys detecting the enrichment of hnRNPL at the promoter of PIK3CB upon paPIK3CB was knocked down. n Representative images and quantitative analysis of hnRNPL puncta in ovarian cancer cells upon paPIK3CB was knocked down by ASOs (n = 50 cells for each group). Scale bar, 5 µm. o Representative images and quantitative analysis of hnRNPL puncta in ovarian cancer cells with RNase treatment (n = 50 cells for each group). Scale bar, 5 µm. p Droplet formation of hnRNPL-EGFP under the influence of different concentrations of CY3-labeled RNA. Scale bar, 50 µm. q Analysis of fluorescent area of EGFP and CY3 signal in each image (n = 3 independent experiments). Data are shown as means ± S.D. p value was calculated by one-way ANOVA test with multiple comparisons ( a, i, n ) and unpaired two-sided Student’s t -test ( e, h, k, m, o ). Data are representative of at least three independent experiments ( a, b, d –f, h, k–p ). Source data are provided as a Source Data file.

Article Snippet: The sequences of oligonucleotides were shown in Supplementary materials. hnRNPL-WT, hnRNPL-IDR1 delG , hnRNPL-IDR2 delP , and hnRNPL-IDR2 FUS expression plasmids were constructed via cloning the corresponding sequence into pCDH-GFP-puro-3×flag, pcDNA3.1-mCherry, and pcDNA3.1-3×flag vector, respectively. hnRNPL-RRM1-1del, hnRNPL-RRM1-2del, hnRNPL-RRM5del and PIK3CB expression plasmids were constructed via cloning corresponding sequence into pcDNA3.1-3×flag vector, respectively. pHR-mCh-Cry2WT was a gift from Clifford Brangwynne (101221, Addgene; http://n2t.net/addgene:101221 ; RRID: Addgene, 101221). pHR-hnRNPL-IDR2 FUS -mCh-Cry2WT, pHR-hnRNPL-WT-mCh-Cry2WT, pHR-hnRNPL-IDR1 delG -mCh-Cry2WT, pHR-hnRNPL-IDR2 delP -mCh-Cry2WT expression plasmids were constructed via cloning corresponding sequence into pHR-mCh-Cry2WT vector, respectively.

Techniques: Construct, Confocal Microscopy, Over Expression, Immunoprecipitation, Western Blot, Quantitative RT-PCR, RNA Expression, Labeling

Journal: Nature Communications

Article Title: hnRNPL phase separation activates PIK3CB transcription and promotes glycolysis in ovarian cancer

doi: 10.1038/s41467-025-60115-7

Figure Lengend Snippet: In ovarian cancer cells, a non-coding RNA transcribed from the PIK3CB promoter recruits hnRNPL and promotes its condensation, which activates PIK3CB transcription and downstream AKT signaling pathway, thus promoting glycolysis and cancer progression.

Article Snippet: The sequences of oligonucleotides were shown in Supplementary materials. hnRNPL-WT, hnRNPL-IDR1 delG , hnRNPL-IDR2 delP , and hnRNPL-IDR2 FUS expression plasmids were constructed via cloning the corresponding sequence into pCDH-GFP-puro-3×flag, pcDNA3.1-mCherry, and pcDNA3.1-3×flag vector, respectively. hnRNPL-RRM1-1del, hnRNPL-RRM1-2del, hnRNPL-RRM5del and PIK3CB expression plasmids were constructed via cloning corresponding sequence into pcDNA3.1-3×flag vector, respectively. pHR-mCh-Cry2WT was a gift from Clifford Brangwynne (101221, Addgene; http://n2t.net/addgene:101221 ; RRID: Addgene, 101221). pHR-hnRNPL-IDR2 FUS -mCh-Cry2WT, pHR-hnRNPL-WT-mCh-Cry2WT, pHR-hnRNPL-IDR1 delG -mCh-Cry2WT, pHR-hnRNPL-IDR2 delP -mCh-Cry2WT expression plasmids were constructed via cloning corresponding sequence into pHR-mCh-Cry2WT vector, respectively.

Techniques: