2026· Methods in molecular biology· Vol 3041, pp.
33-45
· 0 citations
Medicine
TL;DR
An improved tool called pSPIN-GG and supporting protocols for simplified CAST-based genome engineering are presented and refinements support accelerated library construction, reduce assembly and screening burden, and expand the accessibility of CAST systems for multiplexed bacterial genome engineering.
Transposons are fundamental genetic elements that have profoundly shaped the architecture of eukaryotic genomes. Yeasts and filamentous fungi have emerged as important chassis organisms for bioingredient production in synthetic biology and metabolic engineering. In this review, we summarise the current understanding and future opportunities in the development of transposon-based tools for genome engineering in these fungal systems. Fungal inverted terminal repeat (ITR) DNA transposons, as well as long terminal repeat (LTR) and non-LTR retrotransposons, can accelerate genomic mutagenesis, facilitating the screening of superior genotypes and phenotypes. CRISPR-associated transposons (CASTs) hold considerable potential for site-specific integration of large transgenes, bypassing the limitations imposed by low homologous recombination (HR) efficiency in non-Saccharomyces hosts. Overall, transposon-based tools represent a valuable and underexplored avenue to accelerate genome engineering and strain development in yeasts and filamentous fungi.
Bingyin Peng, Masahiro Tominaga, Chengqiang Wang et al.· Yeast· 1 citation
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
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.
MORTISE is introduced, a compact Cas6f-based RNA system to repress target bacterial genes without relying on host RNA factors, enables multiplex control across bacteria, and supports pathway balancing when genetic knockouts are unsuitable.
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.
Smriti Srikanth, Fengyi Zheng, Laura M Drepanos et al.· Cell Genomics· 0 citations
Summary Here, we present a CRISPR-Cas9 genome-editing protocol for Saccharomyces cerevisiae that simplifies guide construction and enables rapid guide iteration by replacing restriction/ligation cloning with PCR-based guide installation and seamless Cas9-plasmid recircularization. We describe single-guide RNA (sgRNA) and homology-directed repair (HDR) donor design, guide insertion into a KanMX/G418-selectable Cas9-sgRNA plasmid, plasmid cloning and sequence verification in E. coli, and lithium acetate/polyethylene glycol (LiAc/PEG) co-transformation of yeast with the verified plasmid and HDR donor.
H. Rostamian, Ethan Madden, Frank M. Kaplan et al.· STAR Protocols· 0 citations