Base-Editing-Mediated Artificial Evolution of OsALS1 In Planta to Develop Novel Herbicide-Tolerant Rice Germplasms

Abstract

Recently developed CRISPR-mediated base editors, which enable the generation of numerous nucleotide changes in target genomic regions, have been widely adopted for gene correction and generation of crop germplasms containing important gain-of-function genetic variations. However, to engineer target genes with unknown functional SNPs remains challenging. To address this issue, we present here a base-editing-mediated gene evolution (BEMGE) method, employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA (sgRNA) library tiling the full-length coding region, for developing novel rice germplasms with mutations in any endogenous gene. To this end, OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bombardment-mediated transformation. Four different types of amino acid substitutions in the evolved OsALS1, derived from two sites that have never been targeted by natural or human selection during rice domestication, were identified, conferring varying levels of tolerance to the herbicide bispyribac-sodium. Furthermore, the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing with the corresponding base editor and sgRNA. Collectively, these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.

Applications of CRISPR technology in studying plant-pathogen interactions: overview and perspective

“…CRISPR/Cas9-mediated base editors and prime editors can be employed to perform precise genome editing of SNPs and SNP-typed QTLs easily in cash crops, endowing multiple resistance to pathogens. Recently, the base-editing-mediated gene evolution (BEMGE) method was developed (Kuang et al 2020). This novel crop breeding method can artificially evolve any endogenous gene in planta with a tiled sgRNA library corresponding to the target genomic locus.…”

Genome editing for plant research and crop improvement

“…Saturation mutagenesis of genes encoding herbicide targets is a promising strategy to find new resistant protein variants. A strategy named base-editing-mediated gene evolution has been used to produce completely new herbicide resistance alleles in rice (Kuang et al, 2020). This method uses Cas9n-based cytosine and ABEs as well as a gRNA library tiling the full-length coding region of the targeted gene.…”

CRISPR base editing and prime editing: DSB and template-free editing systems for bacteria and plants

“…The identified mutations produce PSMB5 and BCR- ABL variants that are resistant to widely used cancer treatments like bortezomid and imatinib, respectively. Similarly, BE systems have effectively been used in plants to enable protein evolution, e.g., screening of ACCase variants [ 149 ] and genomic diversification, e.g., accelerated evolution of OsALS1 [ 150 ] with gRNA library. Development of bacterial or plant protein and genetic libraries using UGI absent CBE or PE could provide functionally diverse protein variants with industrial importance ( Fig.…”