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The prime editors (PEs) have shown great promise for precise genome modification. However, their suboptimal efficiencies present a significant technical challenge. Here, by appending a viral exoribonuclease-resistant RNA motif ( xr RNA) to the 3′-extended portion of pegRNAs for their increased resistance against degradation, we develop an upgraded PE platform (xrPE) with substantially enhanced editing efficiencies in multiple cell lines. A pan -target average enhancement of up to 3.1-, 4.5- and 2.5-fold in given cell types is observed for base conversions, small deletions, and small insertions, respectively. Additionally, xrPE exhibits comparable edit:indel ratios and similarly minimal off-target editing as the canonical PE3. Of note, parallel comparison of xrPE to the most recently developed epegRNA-based PE system shows their largely equivalent editing performances. Our study establishes a highly adaptable platform of improved PE that shall have broad implications. The prime editors (PEs) have shown great promise for precise genome modification. Here the authors place a stabilizing viral xrRNA motif to the 3′ of pegRNAs to enhance editing efficiencies.

The RNA-guided CRISPR-associated (Cas) nucleases are versatile tools for genome editing in various organisms. The large sizes of the commonly used Cas9 and Cas12a nucleases restrict their flexibility in therapeutic applications that use the cargo-size-limited adeno-associated virus delivery vehicle. More compact systems would thus offer more therapeutic options and functionality for this field. Here, we report a miniature class 2 type V-F CRISPR-Cas genome-editing system from Acidibacillus sulfuroxidans (AsCas12f1, 422 amino acids). AsCas12f1 is an RNA-guided endonuclease that recognizes 5′ T-rich protospacer adjacent motifs and creates staggered double-stranded breaks to target DNA. We show that AsCas12f1 functions as an effective genome-editing tool in both bacteria and human cells using various delivery methods, including plasmid, ribonucleoprotein and adeno-associated virus. The small size of AsCas12f1 offers advantages for cellular delivery, and characterizations of AsCas12f1 may facilitate engineering more compact genome-manipulation technologies. Miniature CRISPR-AsCas12f1 has been biochemically characterized and engineered as an effective genome-editing tool for bacteria and human cells.

Polysaccharide capsule is the main virulence factor of K . pneumoniae , a major pathogen of bloodstream infections in humans. While more than 80 capsular serotypes have been identified in K . pneumoniae , only several serotypes are frequently identified in invasive infections. It is documented that the capsule enhances bacterial resistance to phagocytosis, antimicrobial peptides and complement deposition under in vitro conditions. However, the precise role of the capsule in the process of K . pneumoniae bloodstream infections remains to be elucidated. Here we show that the capsule promotes K . pneumoniae survival in the bloodstream by protecting bacteria from being captured by liver resident macrophage Kupffer cells (KCs). Our real-time in vivo imaging revealed that blood-borne acapsular K . pneumoniae mutant is rapidly captured and killed by KCs in the liver sinusoids of mice, whereas, to various extents, encapsulated strains bypass the anti-bacterial machinery in a serotype-dependent manner. Using capsule switched strains, we show that certain high-virulence (HV) capsular serotypes completely block KC’s capture, whereas the low-virulence (LV) counterparts confer partial protection against KC’s capture. Moreover, KC’s capture of the LV K . pneumoniae could be in vivo neutralized by free capsular polysaccharides of homologous but not heterologous serotypes, indicating that KCs specifically recognize the LV capsules. Finally, immunization with inactivated K . pneumoniae enables KCs to capture the HV K . pneumoniae . Together, our findings have uncovered that KCs are the major target cells of K . pneumoniae capsule to promote bacterial survival and virulence, which can be reversed by vaccination.

Most low GC Gram-positive bacteria possess an essential walKR two-component system (TCS) for signal transduction involved in regulating cell wall homoeostasis. Despite the well-established intracellular regulatory mechanism, the role of this TCS in extracellular signal recognition and factors that modulate the activity of this TCS remain largely unknown. Here we identify the extracellular receptor of the kinase ‘WalK’ (erWalK) as a key hub for bridging extracellular signal input and intracellular kinase activity modulation in Staphylococcus aureus . Characterization of the crystal structure of erWalK revealed a canonical Per-Arnt-Sim (PAS) domain for signal sensing. Single amino-acid mutation of potential signal-transduction residues resulted in severely impaired function of WalKR. A small molecule derived from structure-based virtual screening against erWalK is capable of selectively activating the walKR TCS. The molecular level characterization of erWalK will not only facilitate exploration of natural signal(s) but also provide a template for rational design of erWalK inhibitors. The WalKR signal transduction system is involved in extracellular signal recognition, but the details of this function are not well established. Here, the authors report the crystal structure of this two-component system alongside the characterisation of a small-molecule activator.

Tigecycline is a last-resort antibiotic that is used to treat severe infections caused by extensively drug-resistant bacteria. tet (X) has been shown to encode a flavin-dependent monooxygenase that modifies tigecycline 1 , 2 . Here, we report two unique mobile tigecycline-resistance genes, tet (X3) and tet (X4), in numerous Enterobacteriaceae and Acinetobacter that were isolated from animals, meat for consumption and humans. Tet(X3) and Tet(X4) inactivate all tetracyclines, including tigecycline and the newly FDA-approved eravacycline and omadacycline. Both tet (X3) and tet (X4) increase (by 64–128-fold) the tigecycline minimal inhibitory concentration values for Escherichia coli , Klebsiella pneumoniae and Acinetobacter baumannii . In addition, both Tet(X3) ( A. baumannii ) and Tet(X4) ( E. coli ) significantly compromise tigecycline in in vivo infection models. Both tet (X3) and tet (X4) are adjacent to insertion sequence IS Vsa3 on their respective conjugative plasmids and confer a mild fitness cost (relative fitness of >0.704). Database mining and retrospective screening analyses confirm that tet (X3) and tet (X4) are globally present in clinical bacteria—even in the same bacteria as bla NDM-1 , resulting in resistance to both tigecycline and carbapenems. Our findings suggest that both the surveillance of tet (X) variants in clinical and animal sectors and the use of tetracyclines in food production require urgent global attention. Mobile tet (X3) and tet (X4) genes are identified on conjugative plasmids in Enterobacteriaceae and Acinetobacter isolated from humans, meat for consumption and animals that confer resistance to tetracyclines, including tigecycline, eravacycline and omadacycline.

Type V CRISPR-Cas12 systems are highly diverse in their functionality and molecular compositions, including miniature Cas12f1 and Cas12n genome editors that provide advantages for efficient in vivo therapeutic delivery due to their small size. In contrast to Cas12f1 nucleases that utilize a homodimer structure for DNA targeting and cleavage with a preference for T- or C-rich PAMs, Cas12n nucleases are likely monomeric proteins and uniquely recognize rare A-rich PAMs. However, the molecular mechanisms behind RNA-guided genome targeting and cleavage by Cas12n remain unclear. Here, we present the cryo-electron microscopy (cryo-EM) structure of Rothia dentocariosa Cas12n (RdCas12n) bound to a single guide RNA (sgRNA) and target DNA, illuminating the intricate molecular architecture of Cas12n and its sgRNA, as well as PAM recognition and nucleic-acid binding mechanisms. Through structural comparisons with other Cas12 nucleases and the ancestral precursor TnpB, we provide insights into the evolutionary significance of Cas12n in the progression from TnpB to various Cas12 nucleases. Additionally, we extensively modify the sgRNA and convert RdCas12n into an effective genome editor in human cells. Our findings enhance the understanding of the evolutionary mechanisms of type V CRISPR-Cas12 systems and offer a molecular foundation for engineering Cas12n genome editors. Hypercompact type V CRISPR-Cas12n nucleases target DNA sequences containing rare A-rich PAMs. Here, authors reveal the cryo-EM structure of RdCas12n, detailing its RNA-guided DNA targeting and evolutionary origins, and engineer it for effective genome editing in human cells.

The emergence of methicillin-resistant Staphylococcus aureus isolates highlights the urgent need to develop more antibiotics. ClpP is a highly conserved protease regulated by ATPases in bacteria and in mitochondria. Aberrant activation of  bacterial ClpP is an alternative method of discovering antibiotics, while it remains difficult to develop selective   Staphylococcus aureus  ClpP activators that can avoid disturbing Homo sapiens ClpP functions. Here, we use a structure-based design to identify ( R )- and ( S )-ZG197 as highly selective Staphylococcus aureus ClpP activators. The key structural elements in Homo sapiens ClpP, particularly W146 and its joint action with the C-terminal motif, significantly contribute to the discrimination of the activators. Our selective activators display wide antibiotic properties towards an array of multidrug-resistant staphylococcal strains in vitro, and demonstrate promising antibiotic efficacy in zebrafish and murine skin infection models. Our findings indicate that the species-specific activators of Staphylococcus aureus ClpP are exciting therapeutic agents to treat staphylococcal infections. The development of selective ClpP activators targeting only the MRSA isolates without interfering with the human variant is currently challenging. Here, the authors report on the structure-based design of enantiomers of ZG197 and identify the discriminator factor between the proteins.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been harnessed as powerful genome editing tools in diverse organisms. However, the off-target effects and the protospacer adjacent motif (PAM) compatibility restrict the therapeutic applications of these systems. Recently, a Streptococcus pyogenes Cas9 (SpCas9) variant, xCas9, was evolved to possess both broad PAM compatibility and high DNA fidelity. Through determination of multiple xCas9 structures, which are all in complex with single-guide RNA (sgRNA) and double-stranded DNA containing different PAM sequences (TGG, CGG, TGA, and TGC), we decipher the molecular mechanisms of the PAM expansion and fidelity enhancement of xCas9. xCas9 follows a unique two-mode PAM recognition mechanism. For non-NGG PAM recognition, xCas9 triggers a notable structural rearrangement in the DNA recognition domains and a rotation in the key PAM-interacting residue R1335; such mechanism has not been observed in the wild-type (WT) SpCas9. For NGG PAM recognition, xCas9 applies a strategy similar to WT SpCas9. Moreover, biochemical and cell-based genome editing experiments pinpointed the critical roles of the E1219V mutation for PAM expansion and the R324L, S409I, and M694I mutations for fidelity enhancement. The molecular-level characterizations of the xCas9 nuclease provide critical insights into the mechanisms of the PAM expansion and fidelity enhancement of xCas9 and could further facilitate the engineering of SpCas9 and other Cas9 orthologs.

Prime editing allows precise installation of any single base substitution and small insertions and deletions without requiring homologous recombination or double-strand DNA breaks in eukaryotic cells. However, the applications in bacteria are hindered and the underlying mechanisms that impede efficient prime editing remain enigmatic. Here, we report the determination of vital cellular factors that affect prime editing in bacteria. Genetic screening of 129 Escherichia coli transposon mutants identified sbcB , a 3ʹ→5ʹ DNA exonuclease, as a key genetic determinant in impeding prime editing in E. coli , combinational deletions of which with two additional 3ʹ→5ʹ DNA exonucleases, xseA and exoX , drastically enhanced the prime editing efficiency by up to 100-fold. Efficient prime editing in wild-type E. coli can be achieved by simultaneously inhibiting the DNA exonucleases via CRISPRi. Our results pave the way for versatile applications of prime editing for bacterial genome engineering. Prime editing in bacteria is currently inefficient. Here the authors report BacPE, a versatile prime editing platform in Escherichia coli that works by inhibiting 3′→5′ DNA exonucleases, highlighting the intrinsic genetic factors that are adverse to efficient prime editing.

The applicability of nuclease-based form of prime editor (PEn) has been hindered by its complexed editing outcomes. A chemical inhibitor against DNA-PK, which mediates the nonhomologous end joining (NHEJ) pathway, was recently shown to promote precise insertions by PEn. Nevertheless, the intrinsic issues of specificity and toxicity for such a chemical approach necessitate development of alternative strategies. Here, we find that co-introduction of PEn and a NHEJ-restraining, 53BP1-inhibitory ubiquitin variant potently drives precise edits via mitigation of unintended edits, framing a high-activity editing platform (uPEn) apparently complementing the canonical PE. Further developments involve exploring the effective configuration of a homologous region-containing pegRNA (HR-pegRNA). Overall, uPEn can empower high-efficiency installation of insertions (38%), deletions (43%) and replacements (52%) in HEK293T cells. When compared with PE3/5max, uPEn demonstrates superior activities for typically refractory base substitutions, and for small-block edits. Collectively, this work establishes a highly efficient PE platform with broad application potential. Strategies to improve the specificity of nuclease-based prime editor (PEn) are needed. Here the authors report a 53BP1-inhibitory ubiquitin variant-assisted PEn platform (uPEn) to inhibit NHEJ and enable precise prime editing for generation of insertions, deletions and replacements.