Tree physiologyMay 7, 2021

Easy and open-source CRISPR method for editing multiple genes in hybrid aspen

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Apr 2, 2026

Abstract

Out of 22 sequenced alleles of the SHORT ROOT (SHR) gene, 21 were mutated using a gene editing strategy.

An efficient gene editing strategy was developed for hybrid poplar utilizing the Golden Gate MoClo cloning system.
Single gene mutants were successfully generated by targeting the SHR gene with two designed guide RNAs.
The approach also enabled the introduction of mutations in two genes simultaneously, targeting YUC4 and PLT1.
Sequencing confirmed biallelic and homozygous knockout mutations, with common mutations including deletions and small insertions/deletions.
Transgenic roots with biallelic mutations for the SHR gene exhibited a lack of a defined endodermal single cell layer.
The system demonstrated high efficiency for generating double mutants, with biallelic mutations detected in multiple transgenic roots.

Simplified

Although the system has been successfully used for crop breeding, its application remains limited in forest trees. Here, we describe an efficient gene editing strategy for hybrid poplar, (Populus tremula × alba INRA clone 717-1B4) based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two single guide RNAs (sgRNAs) were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate the SHORT ROOT (SHR) gene. Moreover, we also tested its efficiency for introducing mutations in two genes simultaneously by expressing one sgRNA targeting a single site of the YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target the LBD12 and LBD4 genes simultaneously, using an independent construct. We generated hairy roots by Agrobacterium rhizogenes-mediated leaf transformation. Sequencing results confirmed the CRISPR/Cas9-mediated mutation in the targeted sites of PtaSHR. Biallelic and homozygous knockout mutations were detected. A deletion spanning both target sites and small insertions/deletions were the most common mutations. Out of the 22 SHR alleles sequenced, 21 were mutated. The phenotype's characterization showed that transgenic roots with biallelic mutations for the SHR gene lacked a defined endodermal single cell layer, suggesting a conserved gene function similar to its homolog in Arabidopsis Arabidopsis thaliana (L.) Heynh. Sequencing results also revealed the high efficiency of the system for generating double mutants. Biallelic mutations for both genes in the yuc4/plt1 and lbd12/lbd4 roots were detected in three (yuc4/plt1) and two (lbd12/lbd4) out of four transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site was the most common mutations. This CRISPR/Cas9 strategy arises as a rapid, simple and standardized gene-editing tool to evaluate the gene role in essential developmental programs such as radial cell differentiation of poplar roots.

Key numbers

36%

Transformation Efficiency

Percentage of transgenic roots identified from 200 hairy roots.

21 of 22

Mutation Rate

Number of mutated alleles identified out of 22 sequenced.

3 of 4

Double Mutant Success

Number of transgenic roots with biallelic mutations for both targeted genes out of four evaluated.

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Easy and open-source CRISPR method for editing multiple genes in hybrid aspen

Abstract

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