C1000 thermal cycler module

LAMP primers were designed using NEB LAMP Primer Design Tool from New England Biolabs (https://lamp.neb.com/) and PrimerExplorer v5 from Eiken Chemical Co. (https://primerexplorer.jp/e/).
BrCas12b variants, single-guide RNA, fluorescence-quencher reporter, and 10X LAMP primers were combined in 1X Warmstart Multi-Purpose/RT-LAMP Master Mix with UDG (New England Biolabs) to a final concentration of 200 nM, 400 nM, 2000 nM, and 1X LAMP primers (200 nM F3/B3, 1600 nM FIP/BIP, and 800 nM LF/LB), respectively, yielding a volume of 22 mL. The activator (dsDNA/RNA) was added to the mixture (25 mL final volume), and the reaction was then transferred to a CFX96 Real-Time PCR system with a C1000 Thermal Cycler module (Bio-rad). Fluorescence measurements were taken every 30 s per cycle for 120 cycles.

For trans-cleavage assays without the RT-LAMP step, BrCas12b and sgRNA activators were combined to a final concentration of 100 nM: 50 nM respectively in 1X NEBuffer 2.1 (New England Biolabs) and incubated at 65°C for 15 min. Fluorescence-based reporter (FQ) and 25 nM activators containing various PAM sequences were added to the reaction mixture containing the Cas12b: sgRNA complex to a final concentration of 250 nM and 1 nM, respectively. The entire reaction was then immediately transferred to a Bio-rad CFX96 Real-Time system with C1000 Thermal Cycler module. The reaction was isothermally incubated at 65°C, and fluorescence measurements were read every 30 s per cycle over 120 cycles.
In the experiment with RT-LAMP, the one-pot BrCas12b detection assay method was utilized. 3 μL of 10 p.m. activators containing various PAM sequences were added to the reaction. Both experiments were carried out in duplicates and repeated twice.

The thermostable BrCas12b variants and sgRNA were combined in 1X NEBuffer 2.1 (New England Biolabs) to a final concentration of 50 nM: 100 nM (sgRNA: BrCas12b) and then transferred to a pre-heated CFX96 Real-Time PCR system with C1000 Thermal Cycler module (Bio-rad) using a temperature gradient setting across each row, ranging from 60°C to 75°C (60°C, 61°C, 63°C, 65.9°C, 69.5°C, 72.5°C, 74.2°C, and 75°C). The ribonucleoprotein complexes were incubated for 10–60 min with an increment of 10 min. A fluorescence-based reporter (FQ) and dsDNA activator were then added to each mixture to a final concentration of 250 nM and 1 nM, respectively. The reactions were isothermally incubated at their corresponding complexation temperature for consistency. Fluorescent measurements were taken every 30 s for 120 cycles on the HEX channel (λ_ex: 525/10 nm, λ_em: 570/10 nm). This experiment was repeated at incubation temperatures of 67°C and 68°C without the gradient setting to cover a better temperature distribution. All experiments were repeated twice.