SpCas9-NG self-targets the sgRNA sequence in plant genome editing

Area of Science:

• * Molecular Biology
• * Plant Biotechnology
• * Genome Editing

Background:

• * The Streptococcus pyogenes Cas9-NG (SpCas9-NG) enzyme recognizes NGN protospacer adjacent motifs, broadening the applications of genome editing.
• * Genome editing technologies are crucial for genetic research and crop improvement.

Purpose of the Study:

• * To investigate the targeting capabilities of SpCas9-NG in transgenic plants.
• * To assess the potential for SpCas9-NG to self-target single-guide RNA (sgRNA) sequences.
• * To develop strategies for mitigating unintended sgRNA generation by SpCas9-NG.

Main Methods:

• * Transgenic plant generation expressing SpCas9-NG.
• * Analysis of SpCas9-NG activity on both genomic DNA and sgRNA sequences.
• * Design and testing of modified sgRNA scaffolds to prevent self-targeting.

Main Results:

• * SpCas9-NG demonstrated efficient targeting of both genomic DNA and its own sgRNA sequence within transfer DNA in transgenic plants.
• * This self-targeting activity potentially increases off-target risks by generating novel sgRNAs.
• * A modified sgRNA scaffold, initiated with a GCCCC sequence, substantially alleviated the self-target effect.

Conclusions:

• * SpCas9-NG exhibits an intrinsic self-targeting capability in plants, posing a potential risk for unintended sgRNA generation.
• * Modifying the sgRNA scaffold, specifically by starting with a GCCCC sequence, effectively suppresses SpCas9-NG self-targeting.
• * This finding is critical for optimizing the safety and precision of SpCas9-NG in plant genome editing applications.