crrnas  (New England Biolabs)


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    New England Biolabs crrnas
    Identification of the highly active <t>crRNAs</t> for LSDV orf068 gene using CRISPR-Cas12a enhanced fluorescence assay. ( A ). Schematic diagram of the position of crRNAs in LSDV orf068 gene. ( B) . Identification of the highly active crRNAs. crRNA1, 2, 3, 4, 5, 6 represent different crRNAs targeting to LSDV orf068 gene. DNA template represents PCR products of orf068 gene fragment. LSDV, lumpy skin disease virus; crRNA, <t>CRISPR</t> <t>RNA;</t> ssDNA activator is a crRNA-complementary ssDNA. Under blue or UV lights, the pictures were captured under blue (470 nM) and UV lights by a smartphone camera and gel imaging system.
    Crrnas, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    crrnas - by Bioz Stars, 2022-10
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    1) Product Images from "Sensitive and Specific Detection of Lumpy Skin Disease Virus in Cattle by CRISPR-Cas12a Fluorescent Assay Coupled with Recombinase Polymerase Amplification"

    Article Title: Sensitive and Specific Detection of Lumpy Skin Disease Virus in Cattle by CRISPR-Cas12a Fluorescent Assay Coupled with Recombinase Polymerase Amplification

    Journal: Genes

    doi: 10.3390/genes13050734

    Identification of the highly active crRNAs for LSDV orf068 gene using CRISPR-Cas12a enhanced fluorescence assay. ( A ). Schematic diagram of the position of crRNAs in LSDV orf068 gene. ( B) . Identification of the highly active crRNAs. crRNA1, 2, 3, 4, 5, 6 represent different crRNAs targeting to LSDV orf068 gene. DNA template represents PCR products of orf068 gene fragment. LSDV, lumpy skin disease virus; crRNA, CRISPR RNA; ssDNA activator is a crRNA-complementary ssDNA. Under blue or UV lights, the pictures were captured under blue (470 nM) and UV lights by a smartphone camera and gel imaging system.
    Figure Legend Snippet: Identification of the highly active crRNAs for LSDV orf068 gene using CRISPR-Cas12a enhanced fluorescence assay. ( A ). Schematic diagram of the position of crRNAs in LSDV orf068 gene. ( B) . Identification of the highly active crRNAs. crRNA1, 2, 3, 4, 5, 6 represent different crRNAs targeting to LSDV orf068 gene. DNA template represents PCR products of orf068 gene fragment. LSDV, lumpy skin disease virus; crRNA, CRISPR RNA; ssDNA activator is a crRNA-complementary ssDNA. Under blue or UV lights, the pictures were captured under blue (470 nM) and UV lights by a smartphone camera and gel imaging system.

    Techniques Used: CRISPR, Fluorescence, Polymerase Chain Reaction, Imaging

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    New England Biolabs monarch rna cleanup kit
    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized <t>pUC19</t> plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The <t>RNA</t> yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.
    Monarch Rna Cleanup Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.

    Journal: bioRxiv

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    doi: 10.1101/2020.09.14.297226

    Figure Lengend Snippet: VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.

    Article Snippet: The transcripts (pUC19-RNA) were then purified with Monarch RNA Cleanup kit.

    Techniques: SDS Page, Purification, Staining, Plasmid Preparation, Sequencing

    Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.

    Journal: bioRxiv

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    doi: 10.1101/815092

    Figure Lengend Snippet: Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.

    Article Snippet: For the 512B transcripts, reactions were cleaned up with the Monarch® RNA Cleanup Kit (New England Biolabs).

    Techniques: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.

    Journal: bioRxiv

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    doi: 10.1101/815092

    Figure Lengend Snippet: Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.

    Article Snippet: For the 512B transcripts, reactions were cleaned up with the Monarch® RNA Cleanup Kit (New England Biolabs).

    Techniques: Nucleic Acid Electrophoresis, In Vitro

    5’-tRF Cys promotes Nucleolin binding to its target transcripts to enhance their stability. A. Quantification of rRNA levels upon inhibition of 5’-tRF Cys by RT-qPCR. B. Representative polysome profiles showing global translation status in 4T1 cells upon inhibition of 5’-tRF Cys . Mono, monosomes. Di, disomes. C. Percentage of Nucleolin peaks in different types of RNAs. RMSK, repeat masked RNAs. D. The number of Nucleolin-bound CLIP peaks in 5’, 3’ untranslated region (UTR) or coding sequencing (CDS) per 10 kb in the mouse genome. E. Cumulative distribution function (CDF) plots of log 2 FC in transcript abundance for all transcripts stratified by whether they were bound by Nucleolin (red) or not (grey). Statistical significance was determined by Kolmogorov–Smirnov (KS) test (P = 4.8e-13). F. Scatter plot comparing log 2 FC in transcript abundance upon inhibition of 5’-tRF Cys with two distinct 5’-tRF Cys antisense LNAs. Statistically significantly changed genes are marked in red. The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. G. Scatter plot comparing log 2 FC in protein abundance and log 2 FC in transcript abundance between 5’-tRF Cys suppressed and control cells for all transcripts stratified by whether their Nucleolin binding is enhanced by 5’-tRF Cys (red) or not (grey). The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. H, I. Representative western blot images of 5’-tRF Cys targets upon suppression of 5’-tRF Cys (H) or depletion of Nucleolin (I). J. Genome browser view of the aligned Nucleolin (Ncl)-CLIP tags (orange), RNA-Seq reads (red) and Ribo-Seq reads (green) within the 5’ UTR of Pafah1b1. The Y axis represents reads per million (RPM). TSS, transcription start site. K. Quantification by dual luciferase assays of the luminescence signals of reporters containing 5’ UTRs from 5’-tRF Cys targets relative to that from the control GAPDH. Statistical significance in A and K was determined by one-tail t-tests with Welch’s correction. ns, not significant. ***, p

    Journal: bioRxiv

    Article Title: A pro-metastatic tRNA fragment drives Nucleolin oligomerization and stabilization of bound metabolic mRNAs

    doi: 10.1101/2021.04.26.441477

    Figure Lengend Snippet: 5’-tRF Cys promotes Nucleolin binding to its target transcripts to enhance their stability. A. Quantification of rRNA levels upon inhibition of 5’-tRF Cys by RT-qPCR. B. Representative polysome profiles showing global translation status in 4T1 cells upon inhibition of 5’-tRF Cys . Mono, monosomes. Di, disomes. C. Percentage of Nucleolin peaks in different types of RNAs. RMSK, repeat masked RNAs. D. The number of Nucleolin-bound CLIP peaks in 5’, 3’ untranslated region (UTR) or coding sequencing (CDS) per 10 kb in the mouse genome. E. Cumulative distribution function (CDF) plots of log 2 FC in transcript abundance for all transcripts stratified by whether they were bound by Nucleolin (red) or not (grey). Statistical significance was determined by Kolmogorov–Smirnov (KS) test (P = 4.8e-13). F. Scatter plot comparing log 2 FC in transcript abundance upon inhibition of 5’-tRF Cys with two distinct 5’-tRF Cys antisense LNAs. Statistically significantly changed genes are marked in red. The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. G. Scatter plot comparing log 2 FC in protein abundance and log 2 FC in transcript abundance between 5’-tRF Cys suppressed and control cells for all transcripts stratified by whether their Nucleolin binding is enhanced by 5’-tRF Cys (red) or not (grey). The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. H, I. Representative western blot images of 5’-tRF Cys targets upon suppression of 5’-tRF Cys (H) or depletion of Nucleolin (I). J. Genome browser view of the aligned Nucleolin (Ncl)-CLIP tags (orange), RNA-Seq reads (red) and Ribo-Seq reads (green) within the 5’ UTR of Pafah1b1. The Y axis represents reads per million (RPM). TSS, transcription start site. K. Quantification by dual luciferase assays of the luminescence signals of reporters containing 5’ UTRs from 5’-tRF Cys targets relative to that from the control GAPDH. Statistical significance in A and K was determined by one-tail t-tests with Welch’s correction. ns, not significant. ***, p

    Article Snippet: After purified with Monarch RNA Cleanup Kit (NEB Biolabs), RNAs were polyadenylated with E. coli poly(A) polymerase (NEB Biolabs), and capped and 2’-O-methylated with Vaccinia Capping System (NEB Biolabs).

    Techniques: Binding Assay, Inhibition, Quantitative RT-PCR, Cross-linking Immunoprecipitation, Sequencing, Western Blot, RNA Sequencing Assay, Luciferase

    5’-tRF Cys promotes complex D assembly and Nucleolin oligomerization. A, B. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (A) or 5’-tRF Cys (B) using increasing amounts of Nucleolin protein. C. Quantification of complex D assembly as a function of Nucleolin concentration using purified Nucleolin protein. Bmax, specific maximum binding. h, Hill coefficient. Kd, equilibrium dissociation constant. D. Representative images of western blots of Nucleolin from Nucleolin IP that was pre-treated with different dilutions of micrococcal nuclease to remove endogenous RNAs before complexes were assembled at 30 °C and crosslinked with ethylene glycol bis (succinimidyl succinate). The number of blue dots represent the inferred number of Nucleolin monomers based on the molecular weight. E, F. Kinetics of Nucleolin complexes assembled from Pafah1b1 (E) or 5’-tRF Cys (F) using Nucleolin IP. See also Figure 5F . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. G, H. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (G) or 5’-tRF Cys (H) using increasing amount of Nucleolin IP. See also Figure 5G . I. Native gel analysis of Nucleolin complexes assembled using Nucleolin IP from Mthfd1l alone, or together with a wild-type (WT) or Nucleolin binding deficient (MUT) 5’-tRF Cys . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. J. Representative western blot of Nucleolin using Nucleolin IP incubated with or without Pafah1b1, or with both Pafah1b1 and 5’-tRF Cy at 30 °C before crosslinking with EGS. See also Figure 5I . K. Top, quantification of the protection provided by different forms of Nucleolin from degradation by a prototypical 5’- > 3’ exonuclease Terminator after conducting the assembly assay at 4 °C or 30 °C to form monomeric Nucleolin (complex A) or oligomeric Nucleolin (complex D) respectively. Bottom, representative image of denaturing PAGE analysis of the exonuclease degradation products.

    Journal: bioRxiv

    Article Title: A pro-metastatic tRNA fragment drives Nucleolin oligomerization and stabilization of bound metabolic mRNAs

    doi: 10.1101/2021.04.26.441477

    Figure Lengend Snippet: 5’-tRF Cys promotes complex D assembly and Nucleolin oligomerization. A, B. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (A) or 5’-tRF Cys (B) using increasing amounts of Nucleolin protein. C. Quantification of complex D assembly as a function of Nucleolin concentration using purified Nucleolin protein. Bmax, specific maximum binding. h, Hill coefficient. Kd, equilibrium dissociation constant. D. Representative images of western blots of Nucleolin from Nucleolin IP that was pre-treated with different dilutions of micrococcal nuclease to remove endogenous RNAs before complexes were assembled at 30 °C and crosslinked with ethylene glycol bis (succinimidyl succinate). The number of blue dots represent the inferred number of Nucleolin monomers based on the molecular weight. E, F. Kinetics of Nucleolin complexes assembled from Pafah1b1 (E) or 5’-tRF Cys (F) using Nucleolin IP. See also Figure 5F . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. G, H. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (G) or 5’-tRF Cys (H) using increasing amount of Nucleolin IP. See also Figure 5G . I. Native gel analysis of Nucleolin complexes assembled using Nucleolin IP from Mthfd1l alone, or together with a wild-type (WT) or Nucleolin binding deficient (MUT) 5’-tRF Cys . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. J. Representative western blot of Nucleolin using Nucleolin IP incubated with or without Pafah1b1, or with both Pafah1b1 and 5’-tRF Cy at 30 °C before crosslinking with EGS. See also Figure 5I . K. Top, quantification of the protection provided by different forms of Nucleolin from degradation by a prototypical 5’- > 3’ exonuclease Terminator after conducting the assembly assay at 4 °C or 30 °C to form monomeric Nucleolin (complex A) or oligomeric Nucleolin (complex D) respectively. Bottom, representative image of denaturing PAGE analysis of the exonuclease degradation products.

    Article Snippet: After purified with Monarch RNA Cleanup Kit (NEB Biolabs), RNAs were polyadenylated with E. coli poly(A) polymerase (NEB Biolabs), and capped and 2’-O-methylated with Vaccinia Capping System (NEB Biolabs).

    Techniques: Concentration Assay, Purification, Binding Assay, Western Blot, Molecular Weight, Incubation, Polyacrylamide Gel Electrophoresis

    Schematic illustration of RADICA. a , The workflow of RADICA sample partitioning on a chip for absolute quantification of nucleic acid targets. Generally, after the DNA/RNA extraction step, different kind of clinical samples can be used for detection and quantification of various targets. The sample mixture containing DNA/cDNA, RPA reagents, and Cas12a-crRNA-FQ probes is distributed randomly into thousands of partitions. In each partition, the DNA is amplified by RPA and detected by Cas12a-crRNA, resulting in a fluorescent signal in the partition. Based on the proportion of positive partitions and on Poisson distribution, the absolute copy number of the nucleic acid target is quantified. b , Illustration of RPA-Cas12a reaction in each positive partition. In each partition containing the target nucleic acid, the primers bind to the target nucleic acid and initiate amplification with the aid of recombinase and DNA polymerase. Because of the strand displacement of DNA polymerase, the exposed crRNA-targeted ssDNA sites are bound by Cas12a-crRNA complexes. Cas12a is then activated and cleaves the nearby FQ reporters to produce a fluorescence readout.

    Journal: medRxiv

    Article Title: A Digital CRISPR-based Method for the Rapid Detection and Absolute Quantification of Viral Nucleic Acids

    doi: 10.1101/2020.11.03.20223602

    Figure Lengend Snippet: Schematic illustration of RADICA. a , The workflow of RADICA sample partitioning on a chip for absolute quantification of nucleic acid targets. Generally, after the DNA/RNA extraction step, different kind of clinical samples can be used for detection and quantification of various targets. The sample mixture containing DNA/cDNA, RPA reagents, and Cas12a-crRNA-FQ probes is distributed randomly into thousands of partitions. In each partition, the DNA is amplified by RPA and detected by Cas12a-crRNA, resulting in a fluorescent signal in the partition. Based on the proportion of positive partitions and on Poisson distribution, the absolute copy number of the nucleic acid target is quantified. b , Illustration of RPA-Cas12a reaction in each positive partition. In each partition containing the target nucleic acid, the primers bind to the target nucleic acid and initiate amplification with the aid of recombinase and DNA polymerase. Because of the strand displacement of DNA polymerase, the exposed crRNA-targeted ssDNA sites are bound by Cas12a-crRNA complexes. Cas12a is then activated and cleaves the nearby FQ reporters to produce a fluorescence readout.

    Article Snippet: The synthesized crRNA was purified using Monarch® RNA Cleanup Kit (New England Biolabs) after treatment with DNase I (RNase-free, New England Biolabs), Thermolabile Exonuclease I (New England Biolabs), and T5 Exonuclease (New England Biolabs).

    Techniques: Chromatin Immunoprecipitation, RNA Extraction, Recombinase Polymerase Amplification, Amplification, Fluorescence