Native Processing of Single Guide RNA Transcripts to Create Catalytic Cas9/Single Guide RNA Complexes in Planta

The present CRISPR/Cas9 gene editing dogma for single guide RNA (sgRNA) delivery is based on the premise that 5'-and 3'-nucleotide overhangs negate Cas9/sgRNA catalytic activity in vivo. This has led to engineering strategies designed to either avoid or remove extraneous nucleotides at the 5' and 3' termini of sgRNAs. Previously, we used a Tobacco mosaic virus viral vector to express both GFP and a sgRNA from a single virus-derived mRNA in Nicotiana benthamiana This vector yielded high levels of GFP and catalytically active sgRNAs. Here, in an effort to understand the biochemical interactions of this result, we used in vitro assays to demonstrate that nucleotide overhangs 5', but not 3', proximal to the sgRNA do in fact inactivate Cas9 catalytic activity at the specified target site. Next we showed that in planta sgRNAs bound to Cas9 are devoid of the expected 5' overhangs transcribed by the virus. Furthermore, when a plant nuclear promoter was used for expression of the GFP-sgRNA fusion transcript, it also produced indels when delivered with Cas9. These results reveal that 5' auto-processing of progenitor sgRNAs occurs natively in plants. Toward a possible mechanism for the perceived auto-processing, we found, using in vitro-generated RNAs and those isolated from plants, that the 5' to 3' exoribonuclease XRN1 can degrade elongated progenitor sgRNAs, whereas the mature sgRNA end products are resistant. Comparisons with other studies suggest that sgRNA auto-processing may be a phenomenon not unique to plants, but present in other eukaryotes as well.