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[Preprint]. 2023 Feb 17:rs.3.rs-2549171. [Version 1] doi: 10.21203/rs.3.rs-2549171/v1

Fig. 1: Cas12a orthologs tolerate short ssDNA activators (6–12 nt) when added in combination.

Fig. 1:

a. Schematic representation of a crRNA-Cas12a complex performing trans-cleavage of ssDNA reporters following the recognition of two split-activators. b-d. Fold change at t=60 minutes of in vitro trans-cleavage assay with Cas12a orthologs (red = LbCas12a, green = AsCas12a, orange = ErCas12a) activated by individual truncated ssDNA activators of length 6–20 nt e-g. Heat maps representing fold change at t=60 minutes of an in vitro trans-cleavage assay activated by combinations of truncated ssDNA activators of different lengths ranging from 6–14 nt in the Pp and Pd regions. The reactions contained 25 nM truncated ssDNA GFP-activators, 60 nM Cas12a, and 120 nM crGFP and were incubated for 60 min at 37°C. Error bars represent SD (n=3). Statistical analysis was performed using a two-tailed t-test where ns = not significant with p > 0.05, and the asterisks (* p ≤ 0.05, ** p ≤ 0.01,*** p ≤ 0.001, and **** p ≤ 0.0001) denote significant differences.