Extended Data Fig. 3: Comparison of RNA targeting by Ba1Cas12a3 and LshCas13a in TXTL.
From: RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity
(a) GFP silencing in TXTL. Plasmids encoding the Cas nuclease, GFP-targeting or non-targeting crRNA, and deGFP are combined in a TXTL reaction, and GFP fluorescence is measured over time. Right: fluorescence time course for Ba1Cas12a3 and LshCas13a. The vertical dashed line represents the time-point when a separate TXTL reaction was stopped for RNA extraction and RT-qPCR. Curves and shaded regions represent the mean ± standard deviation of separately prepared reactions (n = 3 or 4). (b) RT-qPCR analysis of the deGFP mRNA to determine the extent of mRNA cleavage. Left: location of the two primer pairs used for qPCR relative to the target sites for Ba1Cas12a3 and LshCas13a. Reverse transcription was conducted with the respective reverse primer. Fold-reduction was calculated using the non-target condition as the reference. Each dot represents an independent measurement from one TXTL reaction, while bars and error bars represent the mean ± standard deviation from separate reactions (n = 3), with each the average of triplicate technical measurements. One-tailed paired Student’s t-test. Non-significant p-values (p ≥ 0.05) are in gray.
