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

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Plant Biotechnology

Background:

• Current CRISPR/Cas9 gene editing protocols assume 5' and 3' nucleotide overhangs on single guide RNA (sgRNA) inhibit Cas9 activity.
• This assumption has driven strategies to eliminate these terminal nucleotides in sgRNA engineering.
• Previous work showed a viral vector expressing sgRNA in plants yielded active Cas9, contradicting the dogma.

Purpose of the Study:

• To investigate the biochemical basis for active sgRNA function despite potential overhangs observed in plant systems.
• To determine the role of nucleotide overhangs in sgRNA inactivation of Cas9 catalytic activity.
• To elucidate the mechanism of sgRNA processing in plants and its implications for gene editing.

Main Methods:

• In vitro biochemical assays to test the effect of 5' and 3' sgRNA overhangs on Cas9 activity.
• Analysis of sgRNA structure in planta after binding to Cas9.
• Expression of sgRNA using a plant nuclear promoter and assessment of gene editing outcomes (indels).
• In vitro and in planta degradation assays using the exoribonuclease XRN1.

Main Results:

• In vitro assays confirmed that 5' overhangs, but not 3' overhangs, inactivate Cas9.
• sgRNAs bound to Cas9 in plants lacked the predicted 5' overhangs, suggesting in planta processing.
• Nuclear promoter-driven sgRNA expression resulted in gene editing (indels), supporting 5' auto-processing.
• The 5' to 3' exoribonuclease XRN1 degrades precursor sgRNAs, while mature sgRNAs are resistant.

Conclusions:

• Plants possess a native 5' auto-processing mechanism for progenitor sgRNAs, removing inactivating 5' overhangs.
• This auto-processing ensures the production of catalytically active sgRNAs for CRISPR/Cas9 gene editing in plants.
• sgRNA auto-processing may be a conserved mechanism across eukaryotes, not limited to plants.