Bacteriophages are promising alternatives to traditional antimicrobial treatment of bacterial infections. To further increase the potential of phages, efficient engineering methods are needed. This study investigates an approach to phage engineering based on phage rebooting and compares selected methods of assembly and rebooting of phage genomes. GG assembly of phage genomes and subsequent rebooting by cell-free transcription-translation reactions yielded the most efficient phage engineering and allowed production of a proof-of-concept T7 phage library of 1.8 × 107 phages. We obtained 7.5 × 106 different phages, demonstrating generation of large and diverse libraries suitable for high-throughput screening of mutant phenotypes. Implementing versatile and high-throughput phage engineering methods allows vastly accelerated and improved phage engineering, bringing us closer to applying effective phages to treat infections in the clinic.

Integral and interacting membrane proteins (IIMPs) are crucial biomolecules involved in various life processes, yet their characterization has been hindered by difficulties in purifying and labeling them. This study introduces a novel approach combining cell-free transcription-translation with quartz crystal microbalance with dissipation (TXTL-QCMD). This technique allows for the dynamic characterization of IIMP interactions with lipid bilayers without the need for purification or labeling. The method successfully reconstitutes known IIMP-membrane interactions, including the behavior of E. coli division proteins MinCDE. Additionally, it was applied to the Zorya anti-phage system, revealing specific interactions of the proteins ZorA and ZorB with bacterial membranes, while ZorE exhibited free diffusion. Overall, TXTL-QCMD demonstrates significant potential for exploring the diverse roles of IIMPs in biological systems.

Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA’s presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage—all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.

Ligilactobacillus is a diverse genus among lactobacilli with phenotypes that reflect adaptation to various hosts. CRISPR-Cas systems are highly prevalent within lactobacilli, and Ligilactobacillus salivarius, the most abundant species of Ligilactobacillus, possesses both DNA- and RNA-targeting CRISPR-Cas systems. In this study, we explore the presence and functional properties of I-B, I-C, I-E, II-A, and III-A CRISPR-Cas systems in over 500 Ligilactobacillus genomes, emphasizing systems found in L. salivarius. We examined the I-E, II-A, and III-A CRISPR-Cas systems of two L. salivarius strains and observed occurrences of split cas genes and differences in CRISPR RNA maturation in native hosts. This prompted testing of the single Cas9 and multiprotein Cascade and Csm CRISPR-Cas effector complexes in a cell-free context to demonstrate the functionality of these systems. We also predicted self-targeting spacers within L. salivarius CRISPR-Cas systems and found that nearly a third of L. salivarius genomes possess unique self-targeting spacers that generally target elements other than prophages. With these two L. salivarius strains, we performed prophage induction coupled with RNA sequencing and discovered that the prophages residing within these strains are inducible and likely active elements, despite targeting by CRISPR-Cas systems. These findings deepen our comprehension of CRISPR-Cas systems in L. salivarius, further elucidating their relationship with associated prophages and providing a functional basis for the repurposing of these Cas effectors for bacterial manipulation.

Protocol for myTXTL Antibody/DS Kit (v1.0)

Protocol for myTXTL Pro Kit (v1.0)

Cell-free expression of active antibodies and proteins with disulfide bonds. Just add DNA to express analysis-ready proteins in hours!

Kit for industry-leading cell-free protein expression. Just add DNA to express analysis-ready proteins in hours, and avoid in vivo challenges.

The new myTXTL Antibody/DS Kit enables rapid, cell-free expression of antibody constructs.

The myTXTL Pro Kit is intended for expression of proteins that do not require disulfide bonds. It also has the highest yield of our two kits. The Antibody/DS Kit is intended for the expression of proteins that do contain disulfide bonds such as antibodies and enzymes. The yield of the Antibody/DS kit varies with the protein expressed, but for deGFP it is about 30-50% the yield of the Pro Kit. This is still enough protein for most downstream applications.