DataSheet4_Efficient multi-allelic genome editing via CRISPR–Cas9 ribonucleoprotein-based delivery to Brassica napus mesophyll protoplasts.pdf

Canola (Brassica napus L.) is a valuable oilseed crop worldwide. However, trait improvement by breeding has been limited by its low genetic diversity and polyploid genetics. Whilst offering many potential benefits, the application of transgenic technology is challenged by the stringent and expensive regulatory processes associated with the commercialisation of genetically modified organisms, coupled with a prevailing low public acceptance of such modifications. DNA-free genome editing using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–Cas9 ribonucleoproteins (RNPs) offers a promising way to achieve trait improvements without the limitations of transgenic methods. Here, we present a method for DNA-free genome editing via the direct delivery of RNPs to canola mesophyll protoplasts. This method allows high-throughput in vivo testing of the efficacy of gRNA design as part of the transformation process to facilitate the selection of optimal designs prior to the generation of edited events. Of the 525 shoots regenerated via tissue culture from RNP-transfected protoplasts and screened for the presence of mutations in the targeted gene, 62% had one or more mutated target alleles, and 50% had biallelic mutations at both targeted loci. This high editing efficiency compares favourably with similar CRISPR–Cas9 approaches used in other crop plants.

甘蓝型油菜（Brassica napus L.）是全球极具价值的油料作物。然而，由于其遗传多样性较低且为多倍体基因组，传统育种的性状改良工作一直受到限制。尽管转基因技术具备诸多潜在优势，但其应用却面临严苛且高昂的监管流程阻碍——这些流程与转基因生物商业化密切相关，加之公众对这类基因改造普遍接受度较低。利用成簇规律间隔短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR）–Cas9核糖核蛋白（ribonucleoproteins, RNPs）进行的无DNA基因组编辑技术，为摆脱转基因技术的局限实现性状改良提供了极具前景的解决方案。本研究提出一种通过直接向甘蓝型油菜叶肉原生质体递送RNPs以实现无DNA基因组编辑的方法。该方法可作为转化流程的一部分，实现向导RNA（guide RNA, gRNA）设计有效性的高通量体内测试，以便在生成编辑事件前筛选出最优设计方案。在通过组织培养从RNPs转染的原生质体中再生的525株幼苗中，经靶向基因突变检测后发现，62%的幼苗携带至少一个突变的靶等位基因，50%的幼苗在两个靶向位点均呈现双等位基因突变。该编辑效率与其他作物中应用的同类CRISPR–Cas9编辑方法相比表现优异。