Universal CRISPR-based RNA virus detection and inhibition via foundation model

The use of guide RNA for viral inhibition may result in off-target effects that may disrupt the structural integrity of the endogenous transcriptome. To verify our hypothesis, we conducted a virus inhibition experiment with Cas13d system upon JEV. The CRISPR-viva off-target-free module was employed to generate both off-target-free and off-target-prone guide RNA candidates for JEV and then the fine-tuned CRISPR-viva Cas13d inhibition model was used to predict the efficacy of each guide RNA candidate. We selected 5 off-target-prone guide RNAs targeting three genes (SF23, SMIM13 and DST) to form 6 groups: SF23 (targeting SF23), SM25, SM26, and SM27 (all three targeting SMIM13), DS24 (targeting DST), and NC (negative control with JEV only). After that, we assessed the Cas13d-mediated knock-down effect around the off-target loci in the endogenous RNA as well as on-target loci in the viral RNA through sequencing assay. We first measured the viral titer to confirm the existence of JEV in each group and attained the JEV expression level with the sequencing data.