annealing  (Bolle)

Journal: Nucleic Acids Research

Article Title: Functional decoupling of crRNA enables customizable CRISPR diagnostics

doi: 10.1093/nar/gkag189

Figure Lengend Snippet: Establishment of an independent design axis for cis- cleavage control by RNA oligo-mediated thermodynamic modulation. ( A ) Schematic illustration of functional decoupling framework. Created in BioRender, Park, H. ( https://BioRender.com/k89ldxe ). ( B ) Initial thermodynamic state of LbCas12a, AsCas12a, and enAsCas12a crRNAs hybridized with RNA oligos of varying lengths (6, 8, 10, 12, and 14 nt). ( C ) Agarose gel electrophoresis showing cis- cleavage activity of enAsCas12a complexes with RNA oligos of varying lengths (top). M, 50 bp DNA ladder. (−), Cas12a complex without an RNA oligo. No cleavage, reaction without the Cas12a complex used as a negative control. Band intensities of the remained target were quantified, with the no cleavage condition defined as 100%. Cleavage rates were calculated by subtracting the band intensity of each condition from the no cleavage control and plotted as a function of oligo length. Error bars represent mean ± standard deviation ( n = 3) (bottom). ( D ) Trans -cleavage activity of enAsCas12a measured by fluorescence intensity, normalized to the signal in the absence of RNA oligo, and plotted against oligo length. Error bars represent mean ± standard deviation ( n = 3). ( E ) Heat map showing mean fluorescence intensities across three replicates for LbCas12a, AsCas12a, and enAsCas12a complexes. NTC, no-target control; target, PCR products as activator.

Article Snippet: Cas12a complexes were assembled by incubating Cas12a protein with pre-annealed crRNA–RNA oligo duplex at a 1:1 molar ratio in 1× NEBuffer r2.1 (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2, 100 μg/ml recombinant albumin, pH 7.9, at 25°C) at 25°C for 10 min using the same thermal cycler.

Techniques: Control, Functional Assay, Agarose Gel Electrophoresis, Activity Assay, Negative Control, Standard Deviation, Fluorescence

Journal: Nucleic Acids Research

Article Title: Functional decoupling of crRNA enables customizable CRISPR diagnostics

doi: 10.1093/nar/gkag189

Figure Lengend Snippet: Construction of a one-pot CRISPR diagnostic system using the decoupling framework. ( A ) Schematic illustration of a one-pot CRISPR diagnostic system using decoupling framework. Created in BioRender, Park, H. ( https://BioRender.com/z0xnqjr ). ( B ) Schematic showing how RNA oligo influences one-pot reaction. Created in BioRender, Park, H. ( https://BioRender.com/z0xnqjr ). ( C ) Agarose gel electrophoresis of amplicons generated by one-pot reactions (top). enAsCas12a complexes were tested with RNA oligos of varying lengths (6, 8, 10, 12, and 14 nt). M, 50 bp DNA ladder. (−), Cas12a complex without an RNA oligo. No cleavage, one-pot reaction without the Cas12a complex served as a negative control. Plot of band intensities normalized to the no cleavage band intensity, across RNA oligo length. Error bars represent mean ± standard deviation ( n = 3) (bottom). ( D ) Plot of one-pot reaction fluorescence intensities normalized to the signal at the optimal RNA oligo length, across RNA oligo lengths. The enAsCas12a complex was used. NTC, no-target control; Target, PCR products as target. Error bars represent mean ± standard deviation ( n = 3). ( E ) Heat map of mean fluorescence intensities from triplicate one-pot reactions for LbCas12a, AsCas12a, and enAsCas12a complexes. NTC, no-target control; Target, PCR products as target.

Article Snippet: Cas12a complexes were assembled by incubating Cas12a protein with pre-annealed crRNA–RNA oligo duplex at a 1:1 molar ratio in 1× NEBuffer r2.1 (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2, 100 μg/ml recombinant albumin, pH 7.9, at 25°C) at 25°C for 10 min using the same thermal cycler.

Techniques: CRISPR, Diagnostic Assay, Agarose Gel Electrophoresis, Generated, Negative Control, Standard Deviation, Fluorescence, Control

Journal: Nucleic Acids Research

Article Title: Functional decoupling of crRNA enables customizable CRISPR diagnostics

doi: 10.1093/nar/gkag189

Figure Lengend Snippet: Mechanistic investigation of decoupled cleavage control within a one-pot system. ( A ) Schematic illustration of the fluorescence-based assay designed to monitor the displacement of a quencher-labeled oligo from a Cy5-labeled crRNA. Created in BioRender, Park, H. ( https://BioRender.com/equgvdh ). (B–E) Fluorescence measurements showing the displacement of RNA oligo and one-pot reaction activity. Cy5 signal indicates RNA oligo displacement (red), FAM indicates CRISPR-mediated one-pot reaction (green). ( B ) Real-time fluorescence analysis using a 12 nt RNA oligo, comparing NTC and target. Fluorescence was recorded every 1 min for 15 min. ( C ) Endpoint analysis of FAM and Cy5 fluorescence using a 12 nt RNA oligo. P- values were calculated by unpaired two-tailed Student’s t -test (**** P < .0001, *** P < .001, ** P < .01, * P < .05, ns = not significant). ( D ) Endpoint fluorescence intensity across varying target concentrations. ( E ) Endpoint fluorescence intensity across different oligo lengths. All experiments were performed in triplicate; error bars represent mean ± standard deviation ( n = 3).

Article Snippet: Cas12a complexes were assembled by incubating Cas12a protein with pre-annealed crRNA–RNA oligo duplex at a 1:1 molar ratio in 1× NEBuffer r2.1 (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2, 100 μg/ml recombinant albumin, pH 7.9, at 25°C) at 25°C for 10 min using the same thermal cycler.

Techniques: Control, Fluorescence, Labeling, Activity Assay, CRISPR, Two Tailed Test, Standard Deviation