Abstract
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.
Highlights
The CRISPR/Cas9 platform, found natively in Streptococcus pyogenes, has been developed into a diverse set of functional genetic tools, used in gene editing technology (Mali et al 2013; Cong et al 2013) and transcriptional control through gene activation (Gilbert et al.2013) or repression (Qi et al 2013)
The long standing assumption that single guide RNA (sgRNA) delivery requires specific engineering for removal of 5′ and 3′ nucleotide overhanging sequences present in original progenitor transcripts, that are unrelated to the functional sgRNA sequence, has somewhat handicapped the convenient application and perhaps the efficiency of the technology
Genomic DNA from the mgfp5 harboring transgenic N. benthamiana 16c plants served as a template for amplification of mgfp5 and amplicons were subsequently used as the DNA target for in vitro assays
Summary
The CRISPR/Cas platform, found natively in Streptococcus pyogenes, has been developed into a diverse set of functional genetic tools, used in gene editing technology (Mali et al 2013; Cong et al 2013) and transcriptional control through gene activation (Gilbert et al.2013) or repression (Qi et al 2013). All of these technologies rely on the single guide RNA (sgRNA) programmable endonuclease Cas for specificity.
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