一种用于在[具体生物或细胞类型1]和[具体生物或细胞类型2]中递送CRISPR-Cas核糖核蛋白的原生质体系统。

A Protoplast System for CRISPR-Cas Ribonucleoprotein Delivery in and .

作者信息

Marques Barbara M, Sulis Daniel B, Suarez Bethany, Yang Chenmin, Cofre-Vega Carlos, Thomas Robert D, Whitehill Justin G A, Whetten Ross W, Barrangou Rodolphe, Wang Jack P

机构信息

Forest Biotechnology Group, North Carolina State University, Raleigh, NC 27695, USA.

TreeCo, Raleigh, NC 27606, USA.

出版信息

Plants (Basel). 2025 Mar 22;14(7):996. doi: 10.3390/plants14070996.

DOI:10.3390/plants14070996

PMID:40219064

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11990275/

Abstract

Climate change profoundly impacts the health, productivity, and resilience of forest ecosystems and threatens the sustainability of forest products and wood-based industries. Innovations to enhance tree growth, development, and adaptation offer unprecedented opportunities to strengthen ecosystem resilience and mitigate the effects of climate change. Here, we established a method for protoplast isolation, purification, and CRISPR-Cas ribonucleoprotein (RNP) delivery in and as a step towards accelerating the genetic improvement of these coniferous tree species. In this system, purified protoplasts could be isolated from somatic embryos with up to 2 × 10 protoplasts/g of tissue and transfected with proteins and nucleotides, achieving delivery efficiencies up to 13.5%. The delivery of functional RNPs targeting in and in yielded gene editing efficiencies that reached 2.1% and 0.3%, respectively. This demonstration of RNP delivery for DNA-free genome editing in the protoplasts of and illustrates the potential of CRISPR-Cas to enhance the traits of value in ecologically and economically important tree species. The editing system provides a foundation for future efforts to regenerate genome-edited forest trees to improve ecosystem health and natural resource sustainability.

摘要

气候变化对森林生态系统的健康、生产力和恢复力产生深远影响，并威胁到林产品和木材工业的可持续性。增强树木生长、发育和适应能力的创新为加强生态系统恢复力和减轻气候变化影响提供了前所未有的机遇。在此，我们建立了一种在[具体树种1]和[具体树种2]中进行原生质体分离、纯化以及CRISPR-Cas核糖核蛋白（RNP）递送的方法，作为加速这些针叶树种遗传改良的一步。在该系统中，可从体细胞胚中分离出纯化的原生质体，产量高达每克组织2×10[具体数量]个原生质体，并能用蛋白质和核苷酸进行转染，递送效率高达13.5%。靶向[具体基因1]在[具体树种1]中的功能性RNP递送以及靶向[具体基因2]在[具体树种2]中的递送分别产生了达到2.1%和0.3%的基因编辑效率。在[具体树种1]和[具体树种2]原生质体中进行无DNA基因组编辑的RNP递送证明，说明了CRISPR-Cas在增强具有生态和经济重要性的树种中有价值性状方面的潜力。该编辑系统为未来再生基因组编辑林木以改善生态系统健康和自然资源可持续性的努力奠定了基础。