Efficient CRISPR/Cas9-Mediated Gene Editing in an Interspecific Hybrid Poplar With a Highly Heterozygous Genome

Abstract

Although the CRISPR/Cas9 system has been widely used for crop breeding, its application for the genetic improvement of trees has been limited, partly because of the outcrossing nature and substantial genomic heterozygosity of trees. Shanxin yang (Populus davidiana × P. bolleana), is a commercially important poplar clone that is widely grown in northern China. An established transformation protocol for this interspecific hybrid enables researchers to simultaneously investigate the efficiency and specificity of the CRISPR/Cas9-mediated manipulation of a highly heterozygous genome. Using the phytoene desaturase gene (PDS) as an example, we revealed that the CRISPR/ Cas9 system could efficiently edit the Shanxin yang genome. Two sgRNAs were designed and incorporated into a single binary vector containing the Cas9 expression cassette. Among 62 independent transgenic lines, 85.5% exhibited an exclusively albino phenotype, revealing the total loss of PDS function. The Illumina sequencing results confirmed the targeted mutation of PdbPDS homologs induced by CRISPR/Cas9, and small insertions/deletions were the most common mutations. Biallelic and homozygous knockout mutations were detected at both target sites of the T 0 transformants. Off-target activity was detected for sgRNA2 with a frequency of 3.2%. Additionally, the SNP interference of targeting specificity was assessed based on the sequence variation among PdbPDS homologs. A single mismatch at 19-or 10-bp from the PAM was tolerated by the CRISPR/Cas9 system. Therefore, multiple homologous genes were simultaneously edited despite the presence of a mismatch between the sgRNA and the target site. The establishment of a viable CRISPR/Cas9-based strategy for editing the Shanxin yang genome will not only accelerate the breeding process, but may also be relevant for other economically or scientifically important non-model plants species.

Efficient Genome Editing in Populus Using CRISPR/Cas12a

“…In recent years, CRISPR/Cas9 genome editing systems have been applied in poplar trees (Fan et al, 2015;Zhou et al, 2015;Azeez and Busov, 2020;Wang et al, 2020). However, it is unknown whether Cas12a works in woody tree species.…”

Gene editing in tree and clonal crops: progress and challenges

“…1). We do not consider the case for retention of gene editing machinery in commercial plants, although evidence for extremely low rates of off-target (Wan et al 2017;Wang et al 2017;Yang et al 2017;Elorriaga et al 2018;Fan et al 2018;Muhr et al 2018;Ramos-Sánchez et al 2019;An et al 2020;De Meester et al 2020;Della Maggiora 2020;Ding et al 2020;Fellenberg et al 2020;Movahedi et al 2020;Müller et al 2020;Qin et al 2020;Tsai et al 2020;Wang et al 2020;Azeez and Busov 2021), Malus (Osakabe et al 2018;Charrier et al 2019;Dalla Costa et al 2020;Pompili et al 2020;Zhou et al 2020a), Pyrus (Charrier et al 2019), Citrus (Jia et al 2017b;Jia et al 2017a;Peng et al 2017;Jia et al 2019;Zhu et al 2019;Zou et al 2019;Dutt et al 2020;Huang et al 2020;Jia and Wang 2020;Omori et al 2021) Tuteja et al 2012). Another benefit of marker removal is so that elite-generated insertion events can be stacked with further transgenic traits using the same selectable marker.…”

An Improved CRISPR/Cas9 System for Genome Editing in Populus by Using Mannopine Synthase (MAS) Promoter

“…Hybrid poplars have been genetically modified for studying wood formation, including perennial secondary vascular cambium activity and secondary cell wall deposition (Qiu et al, 2019 ). Despite existence of some promising approaches for creating knockout mutants in Populus (Fan et al, 2015 ; Zhou et al, 2015 ; An et al, 2020a , b ; Wang et al, 2020 ), large-scale gene mutational resources are still lacking for poplars, motivating us to build a robust and high-efficient gene editing system for Populus .…”

“…Woody plants with long-life spans and outcrossing mating systems are difficult subjects for traditional mutagenesis methods (Bewg et al, 2018 ). Currently, there are about ten woody plants species that have been successful edited using CRISPR/Cas9 technology: apple (Nishitani et al, 2016 ), citrus (Jia et al, 2017 ), grape (Nakajima et al, 2017 ), cassava (Odipio et al, 2017 ), cacao (Fister et al, 2018 ), coffee (Breitler et al, 2018 ), kiwifruit (Wang et al, 2018 ), parasponia andersonii (Van Zeijl et al, 2018 ), pomegranate (Chang et al, 2019 ), and poplar (Fan et al, 2015 ; Zhou et al, 2015 ; Wang et al, 2020 ). Three kinds of Populus have been edited via CRISPR/Cas9, including Populus tomentosa Carr (Fan et al, 2015 ), 717 ( Populus tremula × P. alba ) (Zhou et al, 2015 ), and Shanxin yang ( Populus davidiana × P. bolleana ) (Wang et al, 2020 ).…”

“…Currently, there are about ten woody plants species that have been successful edited using CRISPR/Cas9 technology: apple (Nishitani et al, 2016 ), citrus (Jia et al, 2017 ), grape (Nakajima et al, 2017 ), cassava (Odipio et al, 2017 ), cacao (Fister et al, 2018 ), coffee (Breitler et al, 2018 ), kiwifruit (Wang et al, 2018 ), parasponia andersonii (Van Zeijl et al, 2018 ), pomegranate (Chang et al, 2019 ), and poplar (Fan et al, 2015 ; Zhou et al, 2015 ; Wang et al, 2020 ). Three kinds of Populus have been edited via CRISPR/Cas9, including Populus tomentosa Carr (Fan et al, 2015 ), 717 ( Populus tremula × P. alba ) (Zhou et al, 2015 ), and Shanxin yang ( Populus davidiana × P. bolleana ) (Wang et al, 2020 ). Extensively cultivated in China and Korea, the hybrid clone 84K ( Populus alba × P. glandulosa ) is a model hybrid for the study wood formation and stress response and also has relatively high rates of transformation compared with other woody plants (Li et al, 2017 ; Qiu et al, 2019 ).…”