Engineering DNA-Targeting CRISPR and CRISPR-Like Effectors: Advances from Rational Design and High-Throughput Screening to AI-Driven Development

Strategies and mechanisms of precision genome engineering: From gene editing to genome writing

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.

Advances in combinatorial CRISPRi screening and applications: Decoding higher-order interactions for next-generation microbial cell factories in synthetic biology.

This 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, and provides a comprehensive guide for decoding complex traits and driving rational designs of next-generation cell factories.

Genome Editing Using Crispr-Cas9 for Genetic Engineering and Nanozyme Technology: A Review

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. 

Optimized parameters for CRISPR-Cas9 interference library design.

CRISPR interference (CRISPRi) is a powerful technology for studying loss-of-function phenotypes, enabling transient and reversible control of gene expression without the introduction of double-stranded DNA breaks. The cost of conducting large-scale CRISPR screens necessitates the selection of effective and specific single-guide RNAs for the design of compact libraries. While several genome-wide CRISPRi-Cas9 libraries have been created, updates to transcript annotations, the generation of higher-resolution chromatin accessibility datasets, and the development of newer on-target prediction models motivate an updated CRISPRi library design approach. Here, we generate large CRISPRi datasets tiling essential and nonessential genes. We compare the performance of multiple KRAB domain systems, develop an updated CRISPRi-specific on-target scoring scheme, and quantitatively characterize off-target effects associated with seed-sequence patterns. We leverage these findings to design an optimized CRISPRi-Cas9 library, Katsano, and validate its performance with genome-wide viability screens.

Targeted Gene Expression Modulation Using CRISPR/dCas9 to Investigate Pathogenic Outcomes in Tomato.

Current advances in mammalian genome modification technology using the CRISPR-Cas9 principle

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.