Programmable modulation of ribosomal frameshifting by mRNA targeting CRISPR-Cas12a system

Figures

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  Figure 1. Cas12a-crRNA complex can specifically stimulate efficient −1 PRF without triggering RNA cleavage in RRL (A) Representative native agarose gel showing the binding of matched mRNA_v1/crRNA_v1 and mRNA_v1/Cas12a-crRNA_v1 complex as well as the unmatched mRNA_v1/Cas12a-crRNA_v4 complex. (B) Representative denaturing agarose gel showing the RNA cleavage assay of Cas12a. (C) Representative urea-PAGE showing the RNase A cleavage assay of crRNA and Cas12a-crRNA. The doublet of crRNA∗ indicates the pre-crRNA (upper band) and mature crRNA (lower band). An additional higher-resolution image is shown in the lower bracket region. The ssDNA is the spike-in control to normalize crRNA purification. (D) Schematic representation of the representative frameshift reporter construct (p2FL-v1). GFP is in the 0-frame while RFP -1-frame with respect to GFP. The −1 PRF is monitored by the appearance of GFP-RFP fusion product. The UUUAAAC slippery sequence is in bold. The 0-reading frame codons are indicated above the sequence; the −1 frame codons are indicated below the sequence. The sequence of the matched crRNA_v1 is shown. (E) Representative SDS-PAGE analysis showing the [35S]methionine-labeled…

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  Figure 2. Both crRNA and Cas12a-crRNA complex trigger ribosome pausing (A) Schematic representation of the ribosome pausing reporter construct (p2FL-v1-RT). The slippery sequence is mutated to a non-slippery sequence (MSS) highlighted in bold. The immediate downstream codon of the non-slippery sequence is mutated to the UAG stop codon (underlined) to construct p2FL-control. This protein product marks the expected position of crRNA or Cas12a-crRNA-induced ribosome pausing product. Refer to the legend in Figure 1 for the rest of the details. (B–D) The mRNAs of indicated constructs were translated in RRL in the presence of [35S]methionine, after 5 min further initiation was halted by the addition of CR-31, and aliquots were removed at various times and analyzed by SDS-PAGE. Lane C shows makers of ribosome pausing at the MSS. Translation reactions were supplemented with 10 pmol crRNA (50-fold molar excess to the mRNA) (C) or 10 pmol Cas12a-crRNA complex (50-fold molar excess to the mRNA) (D). Full-length and pause products are indicated by filled and empty arrowheads, respectively.

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  Figure 3. Determine the optimal spacer length of crRNA- and Cas12a-crRNA-induced −1 PRF (A) Schematic representation of the −1 PRF reporter constructs based on p2FL-v1. The spacer length between the U3A3C slip site (in bold) and the downstream stimulator, crRNA_v1 or Cas12a- CrRNA_v1, is modified from 3 nt to 11 nt. Refer to the legend to Figure 1 for the rest of the details. (B and C) The reporter mRNAs with indicated spacer length were translated in RRL in the presence of [35S]methionine. Translation products were analyzed by SDS-PAGE followed by successive imaging and quantification. In the construct of 7 nt and 9 nt spacer length, the in-frame product (NFS) is the lower band due to their distinct reading frame caused by the designed spacer length. The shifted products (FS) are indicated. Note that, due to the varied linker sequence, the molecular weight of the FS products is slightly different (Table S2). Translation reactions were supplemented with a 100-fold molar excess of crRNA (B) or a 100-fold molar excess of Cas12a-crRNA complex (C). (D) The bar graph shows the −1 PRF efficiency (FS%) in the presence of crRNA (light gray) or Cas12a-crRNA complex (dark gray). Data represents the…

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  Figure 4. The formation of the Cas12a-crRNA complex enhances the capability of the otherwise inefficient crRNA −1 PRF stimulator with higher AU content (A) Schematic representation of the major sequence context of −1 PRF reporter constructs with the matched crRNAs. Refer to the legend in Figure 1 for the rest of the details. (B) The indicated reporter mRNAs supplemented with matched crRNA or Cas12a-crRNA were translated in RRL in the presence of [35S]methionine. Translation products were analyzed by SDS-PAGE followed by successive imaging and quantification. The quantified −1 PRF efficiency of the representative gel is listed below each lane. FS: frameshifted product; NFS: in-frame product. (C) Bar graph shows the relative −1 PRF efficiency (FS%) normalized to the control without adding crRNA or Cas12a-crRNA complex. Data represent the mean ± SEM of four independent replicates. Statistical analysis (paired t test): ns: not significant, ∗p < 0.05, ∗∗p < 0.01.

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  Figure 5. Deformation of SARS-CoV-2 frameshifting pseudoknot by crRNA and Cas12a-crRNA efficiently attenuate −1 PRF efficiency (A) Schematic representation of the frameshift reporter construct harbors SARS-CoV-frameshifting pseudoknot (p2FL-SARS-CoV-2 PK). The depicted secondary structure is based on the model of Bhatt et al.61 S denotes the stem structure. The three stems are further colored in blue (stem 1, S1), purple (stem 2, S2), and orange (stem 3, S3). Refer to the legend in Figure 1 for the rest of the details. (B) Arrowed lines indicate the designed crRNAs, expressed as Roman numerals, targeted regions of SARS-CoV-2 frameshifting pseudoknot. The crRNA targeting region is further denoted in the bracket: SS denotes slippery sequence; S denotes stem structure; the superscript P and D indicate the proximal targeting or distal targeting, respectively. (C and D) The reporter mRNA supplemented with indicated crRNAs (C) or Cas12a-crRNAs (D) were translated in RRL in the presence of [35S]methionine. Translation products were analyzed by SDS-PAGE followed by successive imaging and quantification. The quantified −1 PRF efficiency of the representative gel is listed below each lane. FS:…

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  Figure 6. Cas12a-crRNA attenuates SARS-2 −1 PRF in human cells (A) Representative images of immunofluorescence staining of HEK293T cells transfected with or without assembled Cas12a-crRNA_VIII complex using HA antibody against the C-terminal 3HA tag of recombinant Cas12a. DAPI is applied to stain nuclear DNA. (B) In vitro transcribed and capped dual luciferase reporter68 mRNA was co-transfected with none, crRNA_VIII, and Cas12a-crRNA_VIII for 8 h, followed by the measurement of luciferase activity. The bar graph shows the relative level of −1 PRF suppression normalized to the none co-transfection control. Data represent the mean ± SD of nine independent biological repeats. Statistical analysis (paired t test): ∗∗p < 0.01.