Variation in Mutation Spectra Among CRISPR/Cas9 Mutagenized Poplars

Area of Science:

• Plant Biotechnology
• Molecular Genetics
• Gene Editing Technologies

Background:

• Transgenic trees require reliable containment methods to address regulatory and public concerns.
• The CRISPR/Cas9 system offers a precise tool for targeted gene modification in plants.

Purpose of the Study:

• To utilize CRISPR/Cas9 to generate loss-of-function mutations in poplar genes essential for flowering, aiming to produce reliably infertile transgenic trees.
• To assess the efficiency and specificity of CRISPR/Cas9 in multiplex gene editing in poplar.

Main Methods:

• Designed synthetic guide RNAs (sgRNAs) targeting poplar homologs of floral genes LEAFY (LFY) and AGAMOUS (AG).
• Generated transgenic poplar events using CRISPR/Cas9 and analyzed mutations via Sanger Sequencing.
• Evaluated mutation spectra, indel types, and off-target effects across different sgRNA constructs and poplar clones.

Main Results:

• Achieved high efficiency in generating double-locus knockouts for AG paralogs (up to 95.5%) and mutations in LFY.
• Observed distinct mutation spectra for each sgRNA-gene combination, with large deletions prevalent when using two active sgRNAs.
• Confirmed high specificity, with no mutations detected in control events or potential off-target sequences.

Conclusions:

• CRISPR/Cas9 is a powerful and precise system for generating loss-of-function mutations in poplar.
• This technology can effectively produce reliably infertile transgenic poplars, potentially aiding regulatory and public acceptance.
• The system's efficiency and specificity support its application in developing contained genetically engineered trees.