Two-guide CRISPR tool makes gene knockouts in poplar trees far simpler
Yang G, Yu Y, Vulavala VKR, Dwivedi N, Liu CJ
Crispr
Poplars are among the fastest-growing trees used for biofuels and carbon sequestration, and sharper gene-editing tools mean researchers can more quickly breed trees that store more carbon or resist pests in the forests you walk through.
When scientists want to turn off a gene in a tree to study what it does, tiny edits often aren't enough to fully disable it, and confirming the edit works requires expensive DNA sequencing. This new approach cuts out a bigger piece of DNA using two molecular scissors working together, so a simple gel test under UV light is all you need to see whether the edit succeeded. The method was developed and tested in hybrid poplar, one of the most important tree species for bioenergy and wood research.
Key Findings
Dual-gRNA CRISPR/Cas9 strategy reliably deletes genomic fragments larger than 50 bp, compared to the small indels typical of single-gRNA approaches.
Mutant trees can be identified by conventional PCR and agarose gel electrophoresis, eliminating the need for routine DNA sequencing during screening.
The protocol was validated using the PtFBX230 gene in Populus tremula x P. alba clone INRA 717-1B4 and is designed to be transferable to other loci and related woody species.
chevron_right Technical Summary
Scientists developed a faster, more reliable method to knock out genes in poplar trees using a two-guide CRISPR system that deletes large chunks of DNA instead of making tiny edits, making it much easier to confirm the mutation worked.
Abstract Preview
Original paper
A Dual-gRNA CRISPR/Cas9 System for Efficient Generation of Large Fragment Deletions in Poplar.
CRISPR/Cas9-based genome editing is a powerful approach for functional genomics and bioenergy research in woody plants. However, conventional single guide RNA (gRNA) strategies predominantly genera...
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Populus is a genus of 25–30 species of deciduous flowering plants in the family Salicaceae, native to most of the Northern Hemisphere. English names variously applied to different species include poplar, aspen, and cottonwood.