FIGURE 1.

Comparison of RNA targeting activity of four CRISPR‐Cas13 orthologs using the transient mGFP5 knockdown assays. (a–d) Schematic of the targeting vectors of four Cas13 protein variants and corresponding crRNA structures. The different Cas13 variants are fused with NLS and 3× FLAG or 3× HA tags under the expression of the U4 promoter. gRNA expression was driven by either the AtU6 promoter or the CmYLCV promoter. DR, specific DRs of different Cas13 systems. The right panel represents the designed spacers complementary to the target RNA (mGFP5) sequences. (e–h) GFP monitoring and reverse transcription quantitative PCR analysis of mGFP5 knockdown to evaluate the Cas13‐mediated RNA interference activities in agroinfiltrated Nicotiana benthamiana leaves. Images and leaf samples were collected 4 days after infiltration. NT, vectors with NT spacer. For each Cas13 variant, the knockdown efficiency of each targeting vector is shown relative to the NT vector. Values are shown as mean ± SEM (n = 13–15)