Validate whether the targeting of crRNAs affected the host cell gene expression

We explored whether the targeting of crRNAs affected the host cell gene expression. To do this, we used a type LNP, termed a lipitoid, to transiently transfect human A549 lung carcinoma cells with the Cas13d mRNA and in vitro synthesized crRNA(s). At 48 hpi, we isolated cellular RNA and performed whole-cell RNA sequencing (RNA-seq). We observed high reproducibility between biological replicates. We examined the off-target effects of viral-targeting crRNA for SARS-CoV-2 or 229E as well as the NT crRNA by comparing their transcriptome profiles to that of cells expressing Cas13d alone, and we saw highly similar gene expression profiles. Furthermore, co-delivery of Cas13d and two targeting crRNAs showed a very similar transcriptome profile to that of the Cas13d alone. These data suggest that Cas13d and antiviral crRNAs are highly specific, with a minimal impact on host cell transcriptomes. Overall design: mRNA profile of A549 cells after two days Cas13d mRNA and related crRNAs transfection