Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba

Abstract

Although the CRISPR/Cas9 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, based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two sgRNAs were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate 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 YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target LBD12 and LBD4 genes simultaneously, using an independent construction. 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. 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 3 (yuc4/plt1) and 2 (lbd12/lbd4) out of 4 transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site were 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.

Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation

“…Multiple mutations in one or both alleles of the same gene can be introduced in an individual plant or hairy root, and this chimerism can be detected only by sequencing. Examples have been described for both stable [ 300 , 301 ] and hairy root [ 302 , 303 ] transformation.…”

CRISPR/Cas9-Mediated Targeted Mutagenesis of FtMYB45 Promotes Flavonoid Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum)

“…The CRISPR/Cas9 gene editing is a fast, simple, efficient, and flexible technique for gene function analysis and crop improvement ( Gupta et al, 2019 ; Triozzi et al, 2021 ). It has been widely used in a variety of plants ( Jinek et al, 2012 ; Feng et al, 2013 ), and has also been applied in medicinal plants ( Li et al, 2017 ; Feng et al, 2018 , 2021 ).…”

CRISPR/Cas9 System: A Potential Tool for Genetic Improvement in Floricultural Crops

“…The most common method of administering gene-editing reagents is through Agrobacterium , which has a narrow host range, and some plant species are resistant to transformation by Agrobacterium . The use of A. rhizogenes , which can substantially minimize the time between reagent administration and mutation evaluation, as well as expand the spectrum of species changed are two further ways to circumvent the regeneration process [ 92 , 144 , 145 ].…”