<scp>RNA</scp> ‐Guided Engineering of the Chloroplast Genome Enabled by Plastid‐Expressed Guide <scp>RNAs</scp>

Our goal is to develop RNA-guided engineering of the chloroplast genome using the CRISPR/Cas9 system. We designed chloroplast minigenes to obtain properly sized single guide RNAs (sgRNAs) in tobacco chloroplasts. The sgRNA 5' end is defined by transcription from an rRNA operon promoter, and its 3' end by processing a downstream tRNA (trnG) or a hepatitis delta virus (HDV) ribozyme. Cas9 is expressed from a nuclear gene and is targeted to chloroplasts by fusion to a transit peptide. Cas9 incorporated the sgRNA and introduced double-strand breaks in the plastid DNA (ptDNA). We report here that the double-strand DNA break in the ndhA and rpoC1 genes was repaired by microhomology-mediated end joining (MMEJ), resulting in deletions in the ptDNA. We further showed that nuclear-expressed sgRNA can be delivered into chloroplasts by fusion with a viroid RNA, as one possible approach for RNA-guided engineering of the ptDNA without direct chloroplast genome transformation. These results are the first step of RNA-guided editing of the chloroplast genome in any crop.