Genomic manipulation has advanced from stochastic nuclease‐mediated disruption toward programmable, deterministic precision. Early clustered regularly interspaced short palindromic repeats (CRISPR) strategies enabled targeted mutagenesis through double‐strand breaks; however, their therapeutic application is limited by genotoxicity, chromosomal instability, and dependence on endogenous repair pathways that are difficult to predict. In this review, we examined the transition from gene editing to genome writing, an approach that decouples genomic modification from host repair pathways to better balance efficiency, precision, and payload delivery. We also discussed the principles of precision technologies, including base and prime editors, and described emerging large‐scale writers, such as CRISPR‐associated transposases and recombinase‐based bridge RNAs, which enable the integration of multi‐kilobase synthetic modules. Beyond enzymatic mechanisms, we further considered the combined use of generative artificial intelligence, structural biology, and novel delivery architectures as potential strategies to overcome current biological limitations. Taken together, these developments point toward Generative Biology, in which computational design and high‐throughput screening transform the genome from a static substrate into a more dynamic model for complex, synthetic functional design.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute adaptive immune systems in prokaryotes and have transformed life sciences, precision medicine, and synthetic biology as programmable genome-editing tools. Despite their broad utility, naturally occurring DNA-targeting Cas effectors remain constrained by several intrinsic limitations, including large protein size that complicates delivery, stringent protospacer adjacent motif (PAM) requirements that restrict targetable genomic space, and mismatch tolerance that can lead to off-target activity and potential genotoxicity. These challenges have made Cas protein engineering and the discovery of novel CRISPR and CRISPR-like systems from metagenomic resources central to the development of next-generation genome-editing platforms. This Review places recent advances within an integrated synthetic biology engineering continuum that links natural effector discovery, structure-guided hypothesis generation, high-throughput functional screening, machine learning-enabled model construction, and iterative redesign. This Review summarizes progress in the screening, optimization, and functional engineering of DNA-targeting CRISPR and CRISPR-like effectors, with emphasis on structure-guided rational design, directed evolution coupled with high-throughput screening, bioinformatics- and evolution-guided mining of novel systems from large-scale sequence databases, and artificial intelligence-assisted development. By integrating these strategies, we highlight how CRISPR effector engineering is moving toward design-build-test-learn (DBTL)-inspired workflows that expand the functional landscape of genome-editing technologies and advance genome editing toward improved efficiency, safety, and programmability.
Lingwei She, Zeyu Liang, Qin Zou et al.· ACS Synthetic Biology· 0 citations
An RNA-guided bridge recombinase system is engineered through rational mutagenesis and AI-assisted directed evolution, enabling programmable chromosomal rearrangements in both plant and mammalian cells and achieving up to a 29.8-fold increase in activity.
Rui Gao, Jingjing Wei, Chao Sun et al.· Trends in Biotechnology· 0 citations
Over the past decade, genome editing has been transformed by RNA‑guided CRISPR‑Cas systems,
which have enabled increasingly precise and programmable DNA modification. Since CRISPR‑Cas9 was
repurposed for genome editing in 2012, base editing (2016) and prime editing (2019) have expanded
the field beyond double‑strand break–dependent repair. Two classes of DNA base-editors have been
developed, cytosine base-editors (CBEs) and adenine base-editors (ABEs). Recently, prime editing has
further expanded the CRISPR editing toolkit to all twelve possible transition and transversion mutations,
as well as small insertion or deletion mutations. Base editors enable precise transition mutations without
double-strand breaks (DSBs). In HEK293T cells, CBE3 has demonstrated a 2‑ to 6‑fold improvement
over CBE2 while maintaining low indel formation (~1.1%). Advances in ABE development include
seventh‑generation variants achieving ~50% efficiency with ≥99.9% product purity and minimal indels
in human cells. In HEK293T cells, third-generation prime editors (PE3) achieved editing efficiencies
of up to ~55%, while the subsequent PE3b strategy reduced indel formation by up to 13-fold without
compromising editing efficiency. CRISPR-Cas9 has achieved clinical success in the treatment of sickle
cell disease and β-thalassemia, while base editing and prime editing have shown promising preclinical
potential for disorders such as cystic fibrosis and Tay-Sachs disease. Together, these findings highlight
the rapid progress of genome editing while underscoring remaining challenges in delivery, specificity,
and clinical translation. This review compares Cas9-mediated homology-directed repair (HDR) with
generations of cytosine base editors (CBE1–CBE3), adenine base editors (ABE1-ABE7), and prime
editors (PE1–PE3b), focusing on their mechanistic distinctions, efficiencies, delivery challenges, and
therapeutic applications.
Anoushka Sinha· American Journal of Student...· 0 citations
This article synthesizes contemporary advancements in CRISPR-mediated mammalian genome modification, detailing core mechanisms – such as guide RNA and the Cas9 endonuclease – alongside next-generation modalities, including base and prime editing.
Olga Aldoshina, Dmitriy Lazarev, E. Smirnova· Veterinariya, Zootekhniya i...· 0 citations
Biological systems possess high robustness and intricate genetic redundancy, rendering traditional single-gene perturbations largely inadequate for comprehensively elucidating the true regulatory mechanisms underlying complex phenotypes. To address this, combinatorial CRISPR interference (CRISPRi) has emerged as an essential tool in systems and synthetic biology, offering reversible epigenetic control, multi-target regulation, and an absence of DNA toxicity. Unlike pooled single-gene screens, combinatorial CRISPRi facilitates the systematic dissection of buffering, synergy, and metabolic trade-offs by targeting multiple loci simultaneously. This review explores the latest advancements in pairwise and higher-order combinatorial CRISPRi screening, beginning with design strategies for multiplex guide RNA (gRNA) arrays and orthogonal systems. Computational techniques utilized for analyzing high-dimensional screening data and visualizing complex genetic networks are subsequently examined. Building upon these methodological foundations, crucial applications within microbial engineering are highlighted. Specifically, the review details the optimization of carbon flux in microbial cell factories to circumvent production bottlenecks, alongside the elucidation of protective multigenic networks against severe environmental stress. Furthermore, it addresses current obstacles, such as system noise and library construction challenges, and outlines future research directions. Ultimately, this review provides a comprehensive guide for decoding complex traits and driving rational designs of next-generation cell factories.
Xiaofei Zhu, Weiwen Zhang, Tao Sun et al.· Microbiology Research· 0 citations
The convergence of CRISPR-Cas9 genome editing and nanozyme engineering is revolutionizing synthetic biology, biotechnology, and medical science. CRISPR-Cas9, a precise and programmable tool, enables targeted genetic modifications that enhance nanozyme functionality, stability, and catalytic efficiency. Through the site-specific mutagenesis, metabolic pathway regulation, and synthetic biology strategies, researchers have significantly improved nanozyme performance for diverse applications, including environmental remediation, biomedical diagnostics, and industrial catalysis. This article explores the fundamental mechanisms of CRISPR-based genome editing, its role in nanozyme optimization, and the latest breakthroughs in enzyme engineering. It also critically examines challenges such as off-target effects, biosafety concerns, and ethical implications associated with gene-edited nanozymes. As advancements in AI-driven predictive modeling and next-generation gene-editing tools emerge, CRISPR-Cas9 is poised to unlock unprecedented possibilities in bioengineering. The integration of genetic precision with catalytic innovation marks a transformative era, redefining the frontiers of molecular biotechnology and paving the way for groundbreaking applications in medicine, industry, and sustainable technology.
Modamori I.O, Okanlawon T.S, Ebhomienlen J.O et al.· Biological and Environmental...· 0 citations