Abstract
Genome editing with sequence-specific nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is revolutionizing crop improvement. Developing efficient genome-editing protocols for highly polyploid crops, including sugarcane (x = 10–13), remains challenging due to the high level of genetic redundancy in these plants. Here, we report the efficient multiallelic editing of magnesium chelatase subunit I (MgCh) in sugarcane. Magnesium chelatase is a key enzyme for chlorophyll biosynthesis. CRISPR/Cas9-mediated targeted co-mutagenesis of 49 copies/alleles of magnesium chelatase was confirmed via Sanger sequencing of cloned PCR amplicons. This resulted in severely reduced chlorophyll contents, which was scorable at the time of plant regeneration in the tissue culture. Heat treatment following the delivery of genome editing reagents elevated the editing frequency 2-fold and drastically promoted co-editing of multiple alleles, which proved necessary to create a phenotype that was visibly distinguishable from the wild type. Despite their yellow leaf color, the edited plants were established well in the soil and did not show noticeable growth retardation. This approach will facilitate the establishment of genome editing protocols for recalcitrant crops and support further optimization, including the evaluation of alternative RNA-guided nucleases to overcome the limitations of the protospacer adjacent motif (PAM) site or to develop novel delivery strategies for genome editing reagents.
Highlights
The processing of sugarcane (Saccharum spp. hybrid) biomass provides 80% of the sugar and 26% of the ethanol produced globally
We explored whether targeted mutagenesis of magnesium chelatase subunit I with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) provides a rapidly scorable phenotype for predicting the extent of multiallelic editing in highly polyploid sugarcane
Events that feature the depletion of chlorophyll are visually distinguishable as soon as the plantlets regenerate from tissue culture (Figure 3A)
Summary
The processing of sugarcane (Saccharum spp. hybrid) biomass provides 80% of the sugar and 26% of the ethanol produced globally. Sugarcane is one of the most productive crops under cultivation due to its superior light conversion and efficiencies of water and nitrogen use (Tew and Cobill, 2008; Byrt et al, 2011). Genome editing using sequencespecific nucleases (SSNs) is a powerful approach for the genetic improvement of crops (Zhang et al, 2018) It has great potential for sugarcane and other vegetatively propagated, heterozygous, and polyploid crops (Weeks, 2017) by enabling precision genome modifications in elite varieties while bypassing meiosis. Among the SSNs, RNA-guided nucleases, including CRISPR/Cas, are the most widely used gene editing tools due to their target specificity, efficiency, simplicity of design, multiplexing capacity, and versatility (Chandrasegaran and Carroll, 2016). They have been repurposed to targeted mutagenesis, gene stacking, targeted nucleotide substitutions, chromosomal translocations, transcriptional or translational regulation, and viral interference (Jinek et al, 2012; Shan et al, 2013; Ali et al, 2015; Baltes et al, 2015; Svitashev et al, 2015; Zhang et al, 2018; Huang and Puchta, 2019; Beying et al, 2020; Gao et al, 2020)
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