CasRx repression work

To facilitate screening of cleavable CasRx designs, we use a synthetic system where we use the fluorescent protein mCherry as the output. Since it has been reported that CasRx can cleave non-target RNA, we first determine the regime in which this collateral activity is minimal. Since collateral activity for CasRx depends on target mRNA level,²⁹ we titrate mCherry expression level (the target mRNA) by changing the promoter of the mCherry plasmid (CMVTO or SFFV) and the amount of plasmid transfected. We also measure GFP fluorescence which is expressed on the same transcript as CasRx (EF1a-GFP-T2A-CasRx) to determine the amount of off-target effect (SFig. 2A). Transfecting 50 ng of SFFV-mCherry plasmid resulted in the greatest fold change in mean mCherry fluorescence between control gRNA and mCherry-targeting gRNA and minimal difference between mean GFP fluorescence.

Based on the results from the previous section, we transfect 50 ng of SFFV-mCherry, 200 ng of the EF1a-GFP-T2A-CasRx_variant, and 200 ng of the mCherry-targeting gRNA for the following cleavable Cas13 experiments.

To engineer a cleavable CasRx, we first screen for locations within CasRx to place the TEV cut site. Ideally, we would like the cleavable CasRx to (1) have similar mRNA cleaving activity as wild-type CasRx and (2) be inhibited by the presence of TEV. Since there is no crystal structure for CasRx, we use AlphaFold to predict its structure and find flexible loops that we can replace with the TEV cut site or where we can insert the TEV cut site. We screened 8 constructs, and our best candidate was cleav_CasRx7 which had similar mRNA cleaving activity as WT CasRx7 (Fig. S2B). However, there was not appreciable TEV repression of mRNA cleavage activity. To improve the ability of TEV to repress activity of cleav_CasRx7, we added a TEV-cleavable N-end degron to the construct. Degrons are used to regulate degradation rates in cells. In the absence of TEV, the degron is masked. After TEV cleaves the N-end degron, the degron will be exposed, marking the protein for degradation, and decreasing its half-life. Since the N- end degron is appended to the N-terminal of the protein, it should not interfere much with the activity of cleav_CasRx7. Indeed, this construct (teD-cleav_CasRx7) was more TEV sensitive than cleav_CasRx7 and retained its efficiency (Fig. S2C).

Lastly, we used teD-cleav_CasRx7 in a whole circuit titration. We transfect 50 ng of SFFV-mCherry and varying amounts of the teD-cleav_CasRx7 circuit. We use BFP as a transfection marker and average the mCherry fluorescence for cells that are highly transfected (BFP fluorescence > 10⁸ A.U.) to produce Fig. 2E. Note that in the running average plots (Fig. S2D), the output mCherry fluorescence increases as dosage increases (BFP transfection marker) which is expected. This is because the mCherry gene is not integrated into the cell’s genome and is instead transcribed from a transfected plasmid. Thus, the relationship between mCherry fluorescence and dosage is not the same as in Fig. 2D which is derived from the simulation which assumes that the target gRNA is produced from an integrated gene.