MND-Cas9 Optimises CRISPR-Cas9 Plant Genome Editing for Large Deletions

Scientists have engineered a modified Cas9 system capable of excising larger DNA segments than standard methods, enabling precise manipulation of gene regulation in crops. This advancement resolves a persistent limitation in **CRISPR-Cas9 plant genome editing**, specifically regarding the study of non-coding DNA.

Limitations in CRISPR-Cas9 Plant Genome Editing

Standard editing tools typically induce small insertions or deletions (indels). While effective for breaking protein-coding genes via frameshift mutations, these minor alterations often fail to disrupt regulatory elements. Non-coding RNAs, untranslated regions (UTRs), and promoter sequences generally require extensive sequence removal to ablate function. Consequently, researchers struggle to investigate the vast non-coding portion of plant genomes. To address this, the study authors sought to create a 'bulldozer' rather than a scalpel.

Engineering the MND-Cas9 System

The research team developed 'Multiple Nucleotide Deletion Cas9' (MND-Cas9) by screening four exonucleases—enzymes that degrade DNA ends. The variants RecJ and T5 underperformed, but TREX2 and SbcB showed promise. By fusing these to Cas9, the team created MND-Cas9v1. It functioned correctly, yet the researchers demanded higher efficiency. They subsequently inserted a DNA-binding domain (DBD) between the nuclease and the exonuclease. This upgrade, MND-Cas9v2, anchors the complex near the cut site. The data indicates that this physical tethering produces significantly larger deletions without sacrificing targeting speed. To further broaden utility, the team incorporated PAM-relaxed variants (Cas9-NG and SpG), allowing the system to access a wider array of genomic addresses previously unreachable by standard Cas9.

Mechanism of Action

The system operates through a coordinated attack. Once the Cas9 domain binds and cuts the target DNA, the tethered exonuclease widens the gap by chewing back the exposed ends. The plant cell then repairs the break, cementing a large deletion. This mechanism is distinct from the random, small scars left by conventional editing. It creates a definitive void in the genetic code.

Validating Agricultural Utility

The study demonstrated the tool's efficacy in rice (*Oryza sativa*). First, the team targeted *OsMIR530*, a microRNA gene. The system successfully executed a knockout, resulting in plants with measurably larger seeds. Second, they deleted sequences within the 3' untranslated region (UTR) of *OsGhd2*. Unlike a knockout, this regulatory disruption upregulated the gene's expression. The result was a tangible increase in grain size. These findings suggest that removing regulatory brakes via large deletions creates new avenues for crop improvement. The MND-Cas9 platform provides the necessary reach to interrogate and engineer the regulatory genome.