Gene editing tools promote the development of chloroplast gene engineering

Plant genetic engineering serves as a crucial technology in enhancing crop quality, promoting pharmaceutical product biosynthesis, and changing agricultural practices. While conventional nuclear transgenic systems demonstrate generally stable and efficient transgene expression profiles, infrequent but persistent technical challenges-including gene silencing as well as low or unstable expression-continue to hinder precise genetic manipulation of nuclear genomes. Since the characteristics of maternal inheritance of plastid genome, chloroplast transformation circumvents this limitation and the risk of transgenic ecological pollution is greatly reduced. Although chloroplast gene engineering (CGE) has some unique advantages, it also has its own disadvantages, including low-efficiency transformation, a limited ability to target organelles, and a low number of species that can transform chloroplast genomes. Over the past few years, the establishment of several novel gene editing technologies has offered beneficial tools to solve these issues. This review explores advanced CGE tools (transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats/CRISPR-associated systems, base editors, and prime editors) for sustainable agriculture, focusing on crop yield improvement, accelerated breeding of resistant varieties, enhanced stress tolerance, and optimized growth traits. Additionally, we thoroughly discuss the current challenges in CGE as well as its potential and future development. Moreover, new technologies and tools, such as nanotechnology, designer pentatricopeptide repeat proteins, and aptamers, are also considered with the aim of improving gene targeting and expression levels in CGE, which could potentially promote advances in CGE and extend its utility for different applications. Challenges in implementation and regulatory considerations are also discussed. • CGE toolkit includes CRISPR/Cas, TALEN, base/prime editors. • CGE faces low efficiency, limited organelle targeting, and few transformable species. • Emerging solutions (nanotech, dPPR, aptamers) boost targeting precision.