Article Title: A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern
Figure Lengend Snippet: BrCas12b characterisation and one-pot specificity sensitivity testing. (a) Schematic of binary complex illustrating cleavage pattern of dsDNA target. (b) Temperature-dependent in-vitro cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2·1. The experiment was repeated ( n = 2 biological replicates) with similar results. (c) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62 °C. (d) Differential scanning fluorimetry of apo AacCas12b, apo AapCas12b, and apo BrCas12b and their corresponding Cas12b:sgRNA complexes. (e) Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. (f)–(i) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 min was monitored using HEX-based reporter. Shaded regions represent standard deviation ( n = 3 biological replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at temperature higher than 60 °C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). (j)–(n) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants of concern ( n = 3 biological replicates). (o) – (t) Limit of detection of non-N gene targeting Alpha (B.1.1.7), Beta (B.1.352), Gamma (P1), Delta (B.1.617.2), Omicron (B.1.1.529) and N gene, respectively ( n = 3 biological replicates).
Article Snippet: In short, BrCas12b, sgRNA, and dsDNA activator were combined to a final concentration of 100 nM : 125 nM : 1 nM, respectively in 1x NEBuffer 2·1 (New England Biolabs) and incubated at 62 °C for 30 min. HEX-polyT-Quencher reporter (FQ) at various concentrations (10 nM, 100 nM, 200 nM, 500 nM, 1 µM, and 2 µM) was added to the reaction mixture containing Cas12b trans-activated complex; and the entire reaction was then immediately transferred to a plate reader.
Techniques: In Vitro, Cleavage Assay, Multiplexing, Amplification, Fluorescence, Standard Deviation