Barybin, A. M. | Search bioRxiv

bioRxiv2026

Synthesis of Ultra-Large Fibrous Proteins from Bacteria via a Looped-Translation System

High molecular weight fibrous proteins such as silk, elastin, and collagens, are fundamental for providing shape to macroscopic biological structures, yet their recombinant production remains challenging because of their extreme size and sequence repetitiveness. Here, we report a circular RNA-based ribosome translation platform that enables iterative ribosome synthesis of fibrous proteins through continuously "looped" translation. To promote efficient circularization of repetitive fibrous protein transcripts, we combined a synonymous codon locker sequence strategy with RNA circularization chaperones. Guided by a ribosome traffic model, we further optimized the translation bottlenecks within the circular RNA, substantially improving translation yields. The established looped translation platform is applicable to at least six classes of fibrous proteins and generated products with molecular weight exceeding titin at 3.8 MDa. The synthesized polypeptides were characterized through electron microscopy, bulk material fabrication, and mechanical analysis, demonstrating properties associated with ultra-high molecular weight polypeptides. Finally, we coupled looped translation to secretion through a programmed ribosomal frameshift, enabling export of fibrous protein across cellular membranes in both Escherichia coli and Bacillus subtilis. We envision that the genetic tools presented here could find a range of applications in bioplastics and engineered living materials.

synthetic biology↗

bioRxiv2025

Anti-CRISPR-mediated continuous directed evolution of CRISPR-Cas9 in human cells

Engineering CRISPR-Cas systems for improved or altered function is central to both research and therapeutic applications. Unfortunately most optimization, especially directed evolution in bacterial hosts, fails to capture the functional requirements of the complex mammalian cellular milieu, where activity is usually required. Robust strategies to enable continuous directed evolution of genome-targeting agents directly in human cells remain lacking. Here, we introduce CRISPR-MACE (Mammalian cell-enabled Adenovirus-assisted Continuous Evolution) as a foundational technology to address this need. CRISPR-MACE integrates virus-based continuous evolution with anti-CRISPR-based tunable selection to generate novel Streptococcus pyogenes Cas9 variants with both increased and decreased DNA binding capacity and nearly 1000-fold-enhanced resistance to AcrIIA4, the strongest known inhibitor of SpCas9. Notably, across independent evolution campaigns the same Cas9 gatekeeper mutation reproducibly emerged first, enabling subsequent adaptive steps along two interdependent axes of Cas9 function. In addition to advancing CRISPR technologies, this work establishes key principles and synthetic circuits for continuously evolving CRISPR-Cas systems directly in human cells. SIGNIFICANCE STATEMENTCRISPR technologies are typically engineered in bacteria, even though they must function in the far more complex environment of human cells. This gap has limited the discovery of variants with improved DNA recognition or with resistance to inhibitors that operate differently in mammalian systems. Here we establish CRISPR-MACE, a continuous evolution platform that leverages pressure from anti-CRISPR proteins to select Cas9 variants directly in human cells that have novel functions. Evolved variants show improvements in DNA binding strength and residence time, as well as striking escape from the potent Cas9 inhibitor AcrIIA4. Many anti-CRISPR proteins use distinct mechanisms, so our strategy can drive future continuous evolution campaigns in mammalian cells that expand the functional properties of genome-targeting agents.

synthetic biology↗