Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and sequence-specific RNA biosensors

Fig. 1. Dual-RNA mediated CRISPRa system reveals the programmability of crRNA-tracrRNA hybridization. a Schematics showing the original sgRNA mediated eukaryote-like CRISPRa system (top) and the dual-RNA mediated CRISPRa system (bottom). Green base pairs, the minimum structure required for CRISPR/Cas9 function; blue bases, RNA aptamer BoxB; red bases, the bulge structure and wobble base pairs (marked as black dots). b Circuit design of the crRNA-tracrRNA mediated CRISPRa system. The activator PspFΔHTH::λN22plus is driven by constitutive promoter J2310638. The anhydrotetracycline (aTc), N-(3-oxohexanoyl)-L-homoserine lactone (AHL), and arabinose are the inducers for promoters Ptet, Plux2, PBAD, respectively. c Test of the combination of three components in the crRNA-tracrRNA mediated CRISPRa, and comparison of the function of gRNAs before and after being split. Induction combinations were achieved via presence (+) or absence (−) of inducers. Error bars, mean values +/− s.d. (n = 4). d Design of the library for testing reprogrammed crRNA-tracrRNA hybridized pairs. The blue letter ‘N’ indicates the substituted base pairs. The representation of motifs in the remaining sequences on the bottom…

Fig. 2. Mode of action of reprogrammed crRNA-tracrRNA pairs. a The length of the hybridized region affects the CRISPR function. The diagram on the left depicts the sequences used and the bar chart on the right exhibits the CRISPRa output. The label ‘mis’ indicates the WT crRNA has a mismatched spacer (LEB3) with the target UAS (LEA2). Error bars, mean values +/− s.d. (n = 3). b RNase III is unnecessary for dual-RNA mediated CRISPRa. The label ‘mis’ is the same as in a. Statistical difference was determined by a two-tailed t test: p = 0.0615, t = 2.577. Error bars, mean values +/− s.d. (n = 3). c Saturated mutations of the GA site. The diagram on the left shows location of the GA site. The heat map on the right shows the results. All the genes were induced for ‘On’ state, no induction for the ‘Off’ state. Data normalized by the output level from the ‘GA’ at ‘On’ state. (n = 4). d Saturated mutations of the base guanine in the core hybridized region. The U5 crRNA-tracrRNA pair was used as an original crRNA version, which had been shown as a functional crRNA-tracrRNA pair (see in Fig. 1e). The data was normalized by the output level from the sample with the WT U5 ‘G’ at ‘On’ state. (n = 4).…

Fig. 3. Engineered tracrRNAs repurpose mRNAs into crRNAs and activate CRISPRa. a Schematic showing the design of the mRNA sensor. Three randomly selected RNA sites (red symbol) are represented. The sequences of three sites are shown in grey letters with GA site highlighted in red. b Green (top) and red fluorescent outputs (bottom) of the mRNA-mediated CRISPRa. WT tracrRNA was used as a mis-matched control. Statistical difference was determined by a two-tailed t test: R1, p = 0.0004, t = 11.01, or by a Welch’s t test: R2, p = 0.0070, t = 10.91; R3, p = 0.0032, t = 17.13. Error bars, mean values ± s.d. (n = 3). c Scatter plot shows the linear relationship between the expression levels of RFP and sfGFP. For R2, Equation: GF = 0.08230×RF + 216.5 (GF: green fluorescence, RF: red fluorescence); the coefficient of determination (R2) is 0.9969; p-value <0.0001. For R3, Equation: GF = 0.05937×RF + 282.8, R2 = 0.9925; p-value < 0.0001. Error bars, mean values ± s.d. (n = 3). d 5’ end truncation on tracrRNA improved CRISPR function. 5’-end extended tracrRNA (tracrRNA) or 5’-end truncated tracrRNA (s-tracrRNA) were employed. For the combination of mRNA and tracrRNA-R2, the same data were used as the…

Fig. 4. Hijacking of endogenous mRNA as crRNA to detect arsenic induced transcription. a Schematic showing the sites selected for tracrRNA targeting. The DNA encoding the endogenous arsenic responsive pathway in E. coli was isolated and inserted into a plasmid under Plux2 promoter. In parallel, the four candidate sites were also extracted separately and inserted into the same vector backbone. The red numbers and short lines show positions of the four candidate sites (Ar1-Ar4). b Test of the availability of the candidate sites on the plasmid transcribed mRNA. The mRNA expressed by the Plux2 promoter was bound to different tracrRNA and corresponding promoters. Error bars, mean values +/− s.d. (n = 3). c Test of the availability of candidate sites on the fragmented mRNAs as crRNAs. crRNAs expressed by the Plux2 promoter were bound to corresponding tracrRNAs and promoters. Inductions are the same as in b. Error bars, mean values +/− s.d. (n = 3). d Schematic showing mechanism of hijacking the endogenous mRNA of the arsenic responsive operon of E. coli to activate reporter expression by CRISPRa. Red symbols indicate the Ar2 targeting site in the polycistronic mRNA of the arsenic-related gene…

Fig. 5. Atypical gRNA-activated Transcription Halting Alarm (AGATHA) system. a Schematic showing the design and mechanism of AGATHA sensor. In the box surrounded by the dashed line, the bordered green bar indicates the Broccoli RNA sequence, and the bordered yellow bar indicates the CRISPR target DNA site paired with the target RNA. The bordered dark purple bar indicates PAM, and the bordered light purple bar indicates an anti-Broccoli tail with an inverted repeat sequence of the Broccoli. The two white triangles mark the position where CRISPR/Cas9 cleaves the AGATHA reporter DNA; the borderless lines indicate gRNA molecules. The cartoon below shows the secondary structure of the RNA aptamer predicted by mfold Web Server39. The polygonal star is the fluorogen, which is activated when binding to the Broccoli aptamer. The colors in the RNA structure diagram are consistent to those in the box above, with the pink segment indicating the T7 terminator. b Specificity test of AGATHA. Two different viral RNA fragments (A and B) from the SARS-CoV-2 virus and the corresponding reprogrammed tracrRNAs were used to test the specificity of AGATHA. The T7 in vitro transcription system was used on…