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.

目前用于单向导RNA（sgRNA）递送的CRISPR/Cas9基因编辑法则基于这样的前提：5'-和3'-核苷酸突出端否定Cas9/sgRNA体内催化活性。这导致了旨在避免或去除 sgRNA 5' 和 3' 末端的外来核苷酸的工程策略。此前，我们使用烟草花叶病毒病毒载体从本塞姆氏烟草中的单个病毒衍生的 mRNA 中表达 GFP 和 sgRNA。该载体产生高水平的 GFP 和催化活性 sgRNA。在这里，为了了解这一结果的生化相互作用，我们使用体外测定来证明靠近 sgRNA 的 5'（而不是 3'）核苷酸突出实际上确实使指定靶位点的 Cas9 催化活性失活。接下来，我们证明在植物中与 Cas9 结合的 sgRNA 缺乏病毒转录的预期 5' 突出端。此外，当植物核启动子用于表达 GFP-sgRNA 融合转录本时，当与 Cas9 一起传递时，它也会产生插入缺失。这些结果表明，祖细胞 sgRNA 的 5' 自动加工在植物中天然发生。针对感知自动加工的可能机制，我们发现，使用体外生成的 RNA 和从植物中分离的 RNA，5' 至 3' 外切核糖核酸酶 XRN1 可以降解伸长的祖 sgRNA，而成熟的 sgRNA 最终产物具有抗性。与其他研究的比较表明，sgRNA 自动加工可能不是植物独有的现象，也存在于其他真核生物中。