Singh Rajveer, Kaur Navneet, Praba Umesh Preethi, Kaur Gurwinder, Tanin Mohammad Jafar, Kumar Pankaj, Neelam Kumari, Sandhu Jagdeep Singh, Vikal Yogesh
School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, India.
Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, India.
Front Genet. 2022 Jun 9;13:882836. doi: 10.3389/fgene.2022.882836. eCollection 2022.
As a staple food crop, rice has gained mainstream attention in genome engineering for its genetic improvement. Genome engineering technologies such as transgenic and genome editing have enabled the significant improvement of target traits in relation to various biotic and abiotic aspects as well as nutrition, for which genetic diversity is lacking. In comparison to conventional breeding, genome engineering techniques are more precise and less time-consuming. However, one of the major issues with biotech rice commercialization is the utilization of selectable marker genes (SMGs) in the vector construct, which when incorporated into the genome are considered to pose risks to human health, the environment, and biodiversity, and thus become a matter of regulation. Various conventional strategies (co-transformation, transposon, recombinase systems, and MAT-vector) have been used in rice to avoid or remove the SMG from the developed events. However, the major limitations of these methods are; time-consuming, leftover cryptic sequences in the genome, and there is variable frequency. In contrast to these methods, CRISPR/Cas9-based marker excision, marker-free targeted gene insertion, programmed self-elimination, and RNP-based delivery enable us to generate marker-free engineered rice plants precisely and in less time. Although the CRISPR/Cas9-based SMG-free approaches are in their early stages, further research and their utilization in rice could help to break the regulatory barrier in its commercialization. In the current review, we have discussed the limitations of traditional methods followed by advanced techniques. We have also proposed a hypothesis, "DNA-free marker-less transformation" to overcome the regulatory barriers posed by SMGs.
作为一种主食作物，水稻在基因组工程以实现其遗传改良方面受到了主流关注。转基因和基因组编辑等基因组工程技术已能够在各种生物和非生物方面以及营养方面显著改善目标性状，而这些方面缺乏遗传多样性。与传统育种相比，基因组工程技术更精确且耗时更少。然而，转基因水稻商业化的一个主要问题是载体构建体中选择标记基因（SMGs）的使用，当这些基因整合到基因组中时，被认为会对人类健康、环境和生物多样性构成风险，因此成为一个监管问题。水稻中已采用各种传统策略（共转化、转座子、重组酶系统和MAT载体）来避免或从已培育的事件中去除选择标记基因。然而，这些方法的主要局限性在于：耗时、基因组中残留隐蔽序列以及频率可变。与这些方法相比，基于CRISPR/Cas9的标记切除、无标记靶向基因插入、程序性自我消除和基于核糖核蛋白（RNP）的递送使我们能够精确且在更短时间内生成无标记的工程水稻植株。尽管基于CRISPR/Cas9的无选择标记基因方法尚处于早期阶段，但在水稻中的进一步研究及其应用可能有助于打破其商业化过程中的监管障碍。在当前的综述中，我们讨论了传统方法的局限性以及先进技术。我们还提出了一个假设，即“无DNA无标记转化”，以克服选择标记基因带来的监管障碍。
