Xueli Zhang

Prime editing is a versatile gene editing technology that has undergone significant enhancements since its inception. Currently, a recently reported prime editing system, PE7 combined with MLH1dn, incorporates two auxiliary components designed to enhance editing efficiency: La fused to the reverse transcriptase (RT) and non-recruited MLH1dn. We hypothesized that the expression and recruitment strategies for these proteins could be further optimized. In this study, we first employed the SunTag system to recruit La and MLH1dn, either individually or simultaneously, and found that recruitment, particularly co-recruitment, significantly enhanced prime editing efficiency. Subsequently, we evaluated various expression and recruitment configurations and determined that a PE system utilizing 2∗GCN4-anchored scFv-fused MLH1dn recruitment, while retaining La fused to RT, achieved the highest editing efficiency. Testing across multiple genomic loci demonstrated that this auxiliary factor–based strategy further improved editing efficiency compared with PE7 alone. In summary, by systematically exploring the recruitment modalities of auxiliary factors, we have developed a more efficient prime editing system, providing researchers with a state-of-the-art tool for high-efficiency genome modification.

Succinic acid (SA) is a key platform chemical with broad applications in biodegradable materials, pharmaceuticals, and food additives, and its market demand continues to expand rapidly. Conventional production processes for SA rely heavily on fossil resources, are associated with substantial carbon emissions, and impose significant environmental burdens, rendering them incompatible with global carbon neutrality goals and the ongoing transition toward sustainable, low-carbon development. Microbial biosynthesis, by contrast, utilizes renewable carbon feedstocks under mild operating conditions and exhibits high atom economy, offering a promising alternative to petrochemical routes. In this review, we systematically trace the historical development of bio-based SA, focusing on technological innovations aimed at cost reduction. We summarize the principal metabolic pathways involved in SA biosynthesis, and critically examine advances in native SA-producing rumen bacteria, metabolically engineered bacteria, and yeast chassis systems. Furthermore, we objectively analyze the application of non-food feedstocks in bio-based SA production and highlight that, despite the current lack of economic competitiveness, such feedstocks represent a crucial direction for the future green and sustainable development of bio-based SA. Finally, we analyze the commercialization trajectory and strategic landscape of bio-based SA both globally and in China. This review aims to provide a conceptual framework and theoretical insights to facilitate the transition of green SA biomanufacturing from laboratory research to large-scale industrial implementation. Graphical Abstract

Bacillus subtilis is an important Generally Recognized as Safe (GRAS) chassis widely used for industrial enzyme production, yet systematic improvement of c

Prime editing (PE) enables precise small DNA changes yet often shows modest efficiency in human cells. Recent advances indicate that the activity of the reverse transcriptase (RT) component is a key determinant of PE performance. Here, we adapt a principle from natural polymerases by fusing a polymerase substrate binding protein (PSBP) with RT to enhance PE. In a twelve-member screen, multiple PSBPs increased editing efficiency, with a compact HMG box module, NHP6A, emerging as a representative lead fusion. NHP6A fused to RT significantly increases intended edits across substitutions, insertions, and deletions while indels remain low. The enhancement is broadly compatible with a nicking sgRNA (PE3), transient mismatch repair inhibition via MLH1dn (PE4), and structured 3′ pegRNA extensions (epegRNA). The PSBP module also synergizes with the La protein-assisted pegRNA tail stabilization (the PE7 system) for additional improvements. Using NHP6A together with La, we efficiently installed four clinically relevant alleles with high product purity. Finally, we validate that PSBP fusion can be consolidated into a single component prime editor without loss of activity. These results establish PSBP fusion as a precise route to improve prime editing outcomes and support integrating compact accessory modules into next-generation PE platforms.

Compact RNA‐guided nucleases such as IscB represent an attractive foundation for next‐generation genome editors, yet their application in mammalian cells has been constrained by suboptimal activity. Here, instead of re‐engineering enzymes, we establish a mutant‐initiated, structure‐guided optimization strategy to generate second‐generation high‐activity IscB editors. Using AlphaFold3 to model the engineered IscB*‐ωRNA–DNA complex, we reveal remodeling of the nucleic‐acid‐binding interface induced by activity‐enhancing substitutions. Guided by this predicted structure, we perform a focused mutational scan and identify V367 as an activity hotspot. Saturation mutagenesis at this position yields a single substitution, V367Y (IscB*‐Act), which increases mean editing efficiency by 34% and achieves up to 2.1‐fold improvement across endogenous targets in mammalian cells. Importantly, the V367Y substitution is transferable to an IscB‐based adenine base editor, elevating A‐to‐G conversion by 68% on average and up to 4.46‐fold at individual loci without altering the intrinsic editing window. Targeted off‐target profiling at loci suggests that V367Y does not substantially increase off‐target indels or A‐to‐G conversion. Together, our work demonstrates a practical framework for second‐generation refinement of compact genome editors, bridging deep‐learning‐enabled structural prediction with interpretable protein engineering, and expands the functional potential of miniature IscB systems for both nuclease and base editing applications.

Genome-scale and multigene transcriptional regulation are crucial technologies in metabolic engineering. However, in Escherichia coli, a stable and universal tool for whole-genome transcriptional activation, and an in situ tool for multigene regulation remain lacking. Here, we present CAGER, a versatile clustered regularly interspaced short palindromic repeats-associated transposases (CAST)-mediated gene regulation toolkit. Through rational mutagenesis, we mitigate the intrinsic transcriptional interference in the left end of CAST system derived from Vibrio cholerae. Using promoters or terminators as cargoes, CAGER constructs the genome-wide activation (3272 genes) or termination (3339 genes) libraries, from which new activation or inhibition targets relevant to cellular acetic acid assimilation are identified. Furthermore, with the aid of M13 phage and the promoter library, CAGER facilitates rapid in situ multigene expression diversification. Applied to lycopene synthesis, a library targeting seven genomic sites is constructed within 24 h, achieving a 73.6-fold yield increase. This work highlights the modifiability of CAST elements and broadens CAST’s application in transcriptional regulation.

Cytosine base editors (CBEs) mediate precise C-to-T conversion and hold considerable therapeutic promise, yet their editing function and utility in oncology remain underexplored. Here, we integrate evolutionary scale modeling (ESM) with structure-guided mutagenesis to remodel human APOBEC3F (A3F), yielding a panel of high-performance CBEs. Our high-efficiency A3F-CBEs achieve up to 1.9- and 3.3-fold higher on-target editing within the canonical editing window than A3A- and Anc689-BE4max, respectively. The high-accuracy A3F-CBEs deliver up to 3.0-fold improvement over haA3A-G at the majority of surveyed loci without compromising specificity. To demonstrate therapeutic potential, we deploy a dual-AAV platform packaging A3F-BE4max and dual gRNAs co-targeting KRAS and MYC in pancreatic ductal adenocarcinoma (PDAC) models. It elicits robust oncogene silencing and inhibited PDAC cell proliferation both in vitro and patient-derived organoids (PDOs). In a PDAC mouse model, it markedly suppresses tumor burden and extends survival. Our work establishes ESM-guided A3F-based CBEs as a versatile, precise platform for cancer genetic therapy.

CRISPR-Cas12a-driven nucleic acid diagnostics offer considerable potential for highly specific and rapid detection. However, their practical applications are limited by the necessity for pre-amplification of target DNA to enhance sensitivity. To overcome this limitation, we developed Auto-catalyst, a novel one-pot, amplification-free DNA detection platform employing a two-stage autocatalytic Cas12a cascade. This approach integrates a positive feedback amplification mediated by a circular crRNA-DNA nanostructure with an asymmetric CRISPR reaction driven by split crRNA. Without external amplification, this system detects DNA fragments at concentrations as low as 80 aM within 30 min at room temperature and maintains high specificity, accurately distinguishing single-base mutations down to 1 fM. Clinical validation demonstrated successful detection of pathogenic DNA in cerebrospinal fluid samples from patients with intracranial infections, highlighting its potential for rapid bedside diagnostics essential for timely clinical decision-making. Additionally, Auto-catalyst accurately identified the clinically significant isocitrate dehydrogenase 1 (IDH1) gene R132H mutation from glioma tissue samples. This integrated two-stage autocatalytic Cas12a strategy represents a powerful, convenient, and promising diagnostic tool suitable for point-of-care applications. Graphical abstract

The yeast Cyberlindnera jadinii has long been used in food and feed industries. While it shows strong intracellular protein synthesis, its limited secretion capacity hampers its use as a protein production host. To overcome this, we used the endogenous invertase CjINV1 as a model and applied multidimensional engineering to the tetraploid wild-type strain NBRC0988. This involved multicopy integration of gene expression cassettes into the chromosome via a landing pad strategy, transcriptional enhancement through combinatorial optimization of promoter and terminator elements, and chitin-based cell wall remodeling to facilitate protein release. The engineered strain showed markedly enhanced protein secretion in a 5-L bioreactor, yielding 3456 ± 81 mg/L extracellular protein after 168 h cultivation, with invertase specific activity of 789.3 ± 52.3 U/mg. To our knowledge, this represents the highest reported yield for yeast-based invertase production to date, highlighting C. jadinii as a promising platform for recombinant protein production.