Unlocking non-model organisms with CRISPR-Cas: A roadmap for sustainable biotechnology.

The reliance on model organisms in biotechnology has advanced our understanding of fundamental biology but has failed to capture the complexity of real-world ecosystems, limiting applications in agriculture, biomanufacturing, and environmental remediation. This review critically evaluates the challenges and opportunities of applying CRISPR-Cas genome editing to non-model organisms, structured around a framework that systematically addresses host-specific barriers, enabling technical solutions, and real-world applications. Key obstacles are first delineated, such as restrictive genetic tools, inefficient DNA repair pathways (including NHEJ-dominance, HDR-deficiency, and polyploidy), and delivery limitations. Subsequently, innovative solutions are explored, including the engineering of Cas variants with expanded PAM flexibility and reduced toxicity, the development of host-adapted delivery systems such as phage-based vectors and conjugative plasmids, and the integration of synthetic biology tools and machine learning for optimization. Alternative, DSB-free modalities-comprising base editing, prime editing, CRISPR-associated transposases (CAST), and recombinase-assisted engineering-are further expanded upon, offering enhanced precision and expanded capabilities for complex genetic modifications. Major findings indicate that these approaches can unlock the potential of non-conventional hosts to address global challenges, such as low-energy biomanufacturing, environmental bioremediation, and carbon capture. It is concluded that bridging the gap between foundational CRISPR research and its real-world applications is imperative. Future efforts should focus on democratizing tools via open-source platforms, advancing delivery systems, establishing ethical governance-with detailed considerations for environmental release, horizontal gene transfer, regional regulatory frameworks, and biosafety in extremophile engineering-and fostering sustainable innovation through synthetic biology integration to fully realize the transformative potential of genome editing in organisms beyond model organisms.