CRISPR/ddCas12a-based programmable and accurate gene regulation

a Illustration of the positions of crRNAs designed for repression of gfp transcription. b ddCas12a-mediated repression of gfp transcription in E. coli MG1655. Both the gfp mRNA transcriptional level and the fluorescence intensities were measured, both of which demonstrated the effectiveness of repression by crRNAs targeting to the promoter region or the T strand of gfp. Cells expressing ddCas12a and the empty vector pTC17401 were employed as a control, where the transcriptional level of gfp was normalized to 1000 and the fluorescence intensities were normalized using the OD600 value. c Correlation analysis between the gfp transcription and the gfp fluorescence intensities detected in Fig. 1b. Both data were analyzed using the mean value, with an R2 value of 0.9953. d Illustration of the four mutant libraries with random mutations within the 19-nt crRNA DR sequences. The wild type crRNA DR structure for ddCas12a was shown above, and four mutant libraries (i.e., crRNA-ML1 to crRNA-ML4) were shown below, where “N” in the mutant libraries represented randomized nucleotide substitution on the corresponding position. e Illustrative chart of the library screening using the flow sorting method. The obtained mutant libraries were co-transformed with ddCas12a in E. coli, followed by the flow sorting according to the fluorescence signal intensities. f Quantitative repression of gfp transcription with the screened mutant crRNAs. Totally six mutant crRNAs (i.e., 10–7B, 10–8？C, 11–3？F, 9–2D, 10–7？F and 10–5B) were used for analysis, and two distinct targeting sites (i.e., T1 and T2) on gfp were tested, employing cells expressing ddCas12a and the empty vector pTC17401 as a negative control or ddCas12a and the wild type crRNA as a positive control. The fluorescence intensities were measured by Varioskan Flash and normalized with the OD600 value. g Quantitative repression of DsRed transcription. Mutant crRNA DR sequences were the same as those in Fig. 1f, and the trends of the repression efficiencies by each mutant crRNA were the similar to those of gfp repression. h Analysis of the binding affinities of ddCas12a against mutated crRNAs and target dsDNA. Three mutant crRNAs (i.e., 10–5B, 9–2D and 10–7B), which targeted to the T1 site of gfp and showed different efficiencies for ddCas12a-based repression, were used for EMSA analysis. The ratio of shifted crRNA to total labeled crRNA probes was calculated by the fluorescence intensity and shown below each lane, and the binding affinities were the highest for 10–5B and lowest for 10–7B, which were consistent with those of