Fig. 2: In vitro development of the DNACas strategy.
From: Scalable modulation of CRISPR‒Cas enzyme activity using photocleavable phosphorothioate DNA
a Schematic diagram illustrating the modulation of LbCas12a system activity by photoregulation of PC&PS DNA. b–d Measurement of the light-switching behavior of PC&PS DNA (random base composition, 26 nt in length) embedded with different amounts of PC-linker against trans-cleavage of LbCas12a (b) and LbuCas13a (c), and cis-cleavage of LbCas12a (d). e Measurement of the light-switching behavior of three PC&PS DNAs against LbCas12a. PC&PS DNA1, PC&PS DNA2, and PC&PS DNA3 have the same length (21 bases with 20 PS modifications) and are embedded with the same number of PC-linkers (10 PC-linkers) with different base compositions. f Evaluation of the concentration-dependent inhibitory effects of LbCas12a by three PC&PS DNAs (20PS10PC, 25PS12PC, 36PS18PC) with varying lengths. The experiment was conducted without UV light treatment. The final concentration of the LbCas12a is 15 nM. Relative activity is defined as the ratio of LbCas12a activity in the experimental group containing PC&PS DNA to that in the control group without PC&PS DNA. g The chemical structural formulas of PO DNA, PS DNA, and PC&PS DNA. h, i Evaluation of the photoregulatory ability of PO DNA, PS DNA, and PC&PS DNA on cis-cleavage activity of SpCas9 (h) and trans-cleavage activity of BrCas12b (i). The activity recovery was calculated by comparison to the native Cas protein for cleaving the same target. The long linear dsDNA containing the putative on-target sequence was cleaved by the Cas9 protein complexed with single-guide RNA (sgRNA), resulting in two shorter dsDNA fragments. The cleavage rate in (h) was determined using ImageJ software by measuring the gray-values of the uncleaved band and calculating its relative ratio to the blank (representing blank control containing only the template DNA). The total length of the DNA sequences is equal to the number of PS modifications plus one. These DNA sequences and RNP were preincubated at room temperature for 5 min. In all experiments, UV represents UV (365 nm) light irradiation for 1 min. Data are represented as mean \(\pm\) standard error (n = 3 technical replicates). Source data are provided as a Source Data file.
