BacPE: a versatile prime-editing platform in bacteria by inhibiting DNA exonucleases.

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 3'→5' DNA exonuclease, as a key genetic determinant in impeding prime editing in E. coli, combinational deletions of which with two additional 3'→5' 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.

Molecular Basis and Genome Editing Applications of a Compact Eubacterium ventriosum CRISPR-Cas9 System.

CRISPR-Cas9 systems have been widely harnessed for diverse genome editing applications because of their ease of use and high efficiency. However, the large molecular sizes and strict PAM requirements of commonly used CRISPR-Cas9 systems restrict their broad applications in therapeutics. Here, we report the molecular basis and genome editing applications of a novel compact type II-A Eubacterium ventriosum CRISPR-Cas9 system (EvCas9) with 1107 residues and distinct 5'-NNGDGN-3' (where D represents A, T, or G) PAM specificity. We determine the cryo-EM structure of EvCas9 in a complex with an sgRNA and a target DNA, revealing the detailed PAM recognition and sgRNA and target DNA association mechanisms. Additionally, we demonstrate the robust genome editing capacity of EvCas9 in bacteria and human cells with superior fidelity compared to SaCas9 and SpCas9, and we engineer it to be efficient base editors by fusing a cytidine or adenosine deaminase. Collectively, our results facilitate further understanding of CRISPR-Cas9 working mechanisms and expand the compact CRISPR-Cas9 toolbox.

Molecular basis and engineering of miniature Cas12f with C-rich PAM specificity.

CRISPR-Cas12f nucleases are currently one of the smallest genome editors, exhibiting advantages for efficient delivery via cargo-size-limited adeno-associated virus delivery vehicles. Most characterized Cas12f nucleases recognize similar T-rich protospacer adjacent motifs (PAMs) for DNA targeting, substantially restricting their targeting scope. Here we report the cryogenic electron microscopy structure and engineering of a miniature Clostridium novyi Cas12f1 nuclease (CnCas12f1, 497 amino acids) with rare C-rich PAM specificity. Structural characterizations revealed detailed PAM recognition, asymmetric homodimer formation and single guide RNA (sgRNA) association mechanisms. sgRNA engineering transformed CRISPR-CnCas12f1, which initially was incapable of genome targeting in bacteria, into an effective genome editor in human cells. Our results facilitate further understanding of CRISPR-Cas12f1 working mechanism and expand the mini-CRISPR toolbox.

A KDPG sensor RccR governs Pseudomonas aeruginosa carbon metabolism and aminoglycoside antibiotic tolerance.

Pseudomonas aeruginosa harbors sophisticated transcription factor (TF) networks to coordinately regulate cellular metabolic states for rapidly adapting to changing environments. The extraordinary capacity in fine-tuning the metabolic states enables its success in tolerance to antibiotics and evading host immune defenses. However, the linkage among transcriptional regulation, metabolic states and antibiotic tolerance in P. aeruginosa remains largely unclear. By screening the P. aeruginosa TF mutant library constructed by CRISPR/Cas12k-guided transposase, we identify that rccR (PA5438) is a major genetic determinant in aminoglycoside antibiotic tolerance, the deletion of which substantially enhances bacterial tolerance. We further reveal the inhibitory roles of RccR in pyruvate metabolism (aceE/F) and glyoxylate shunt pathway (aceA and glcB), and overexpression of aceA or glcB enhances bacterial tolerance. Moreover, we identify that 2-keto-3-deoxy-6-phosphogluconate (KDPG) is a signal molecule that directly binds to RccR. Structural analysis of the RccR/KDPG complex reveals the detailed interactions. Substitution of the key residue R152, K270 or R277 with alanine abolishes KDPG sensing by RccR and impairs bacterial growth with glycerol or glucose as the sole carbon source. Collectively, our study unveils the connection between aminoglycoside antibiotic tolerance and RccR-mediated central carbon metabolism regulation in P. aeruginosa, and elucidates the KDPG-sensing mechanism by RccR.

Cas12n nucleases, early evolutionary intermediates of type V CRISPR, comprise a distinct family of miniature genome editors.

Type V CRISPR-associated systems (Cas)12 family nucleases are considered to have evolved from transposon-associated TnpB, and several of these nucleases have been engineered as versatile genome editors. Despite the conserved RNA-guided DNA-cleaving functionality, these Cas12 nucleases differ markedly from the currently identified ancestor TnpB in aspects such as guide RNA origination, effector complex composition, and protospacer adjacent motif (PAM) specificity, suggesting the presence of earlier evolutionary intermediates that could be mined to develop advanced genome manipulation biotechnologies. Using evolutionary and biochemical analyses, we identify that the miniature type V-U4 nuclease (referred to as Cas12n, 400-700 amino acids) is likely the earliest evolutionary intermediate between TnpB and large type V CRISPR systems. We demonstrate that with the exception of CRISPR array emergence, CRISPR-Cas12n shares several similar characteristics with TnpB-ωRNA, including a miniature and likely monomeric nuclease for DNA targeting, origination of guide RNA from nuclease coding sequence, and generation of a small sticky end following DNA cleavage. Cas12n nucleases recognize a unique 5'-AAN PAM sequence, of which the A nucleotide at the -2 position is also required for TnpB. Moreover, we demonstrate the robust genome-editing capacity of Cas12n in bacteria and engineer a highly efficient CRISPR-Cas12n (termed Cas12Pro) with up to 80% indel efficiency in human cells. The engineered Cas12Pro enables base editing in human cells. Our results further expand the understanding regarding type V CRISPR evolutionary mechanisms and enrich the miniature CRISPR toolbox for therapeutic applications.

Using CRISPR-Cas9-Based Methods for Genome Editing in Staphylococcus aureus.

Chromosomal mutations and targeted gene deletions and inactivations in Staphylococcus aureus are typically generated using the allelic exchange method. In recent years, however, more rapid methods have been developed, often using CRISPR-Cas9-based systems. Here, we describe recently developed CRISPR-Cas9-based plasmid systems for use in S. aureus, and discuss their use for targeted gene mutation and inactivation. First, we describe how a CRISPR-Cas9 counterselection strategy can be combined with a recombineering strategy to generate gene deletions in S. aureus We then introduce dead Cas9 (dCas9) and Cas9 nickase (nCas9) enzymes, and discuss how the nCas9 enzyme fused to different nucleoside deaminases can be used to introduce specific base changes in target genes. We then discuss how the nCas9-deaminase fusion enzymes can be used for targeted gene inactivation via the introduction of premature stop codons or by mutating the start codon. Together, these tools highlight the power and potential of CRISPR-Cas9-based methods for genome editing in S. aureus.

Introduction of a CRISPR-nCas9 Gene-Targeting Plasmid into Staphylococcus aureus for Gene Disruption.

Methods for gene disruption are essential for functional genomics, and there are multiple approaches for altering gene function in bacteria. One of these methods involves introducing a premature stop codon in a gene of interest, which can be achieved by using the CRISPR-nCas9-cytidine deaminase system. The approach involves the mutation of editable cytidines to thymidines, with the goal of generating a novel stop codon that ultimately results in a nonfunctional gene product. The workflow involves two major sections, one for the identification of editable cytidines, the design of the targeting spacer oligonucleotides for introduction into the CRISPR-nCas9 cytidine deaminase plasmid, and the construction of the gene-targeting CRISPR-nCas9 cytosine deaminase plasmids, and one for the actual introduction of the mutation in the species of interest. Here, we describe the steps for the second part. Specifically, we describe (1) how to introduce the gene-targeting pnCasSA-BEC plasmid into Staphylococcus aureus, (2) how the gene inactivation in S. aureus can be confirmed by PCR and sequencing, and (3) how, following successful gene inactivation, the strain can be cured of the pnCasSA-BEC plasmid. To better illustrate the method, and as specific example, two different geh gene-inactivation mutations are generated here in S. aureus RN4220. The protocol, however, can easily be adapted to generate other gene-inactivating mutations.

Identification of Editable Sites, Spacer Oligonucleotide Design, and Generation of the Gene-Targeting CRISPR-nCas9 Plasmid for Gene Disruption in Staphylococcus aureus Using the CRISPR-nCas9 and Cytidine Deaminase System.

Methods for gene disruption are essential for functional genomics, and there are multiple approaches for altering gene function in bacteria. One of these methods involves introducing a premature stop codon in a gene of interest, which can be achieved by using the CRISPR-nCas9-cytidine deaminase system. The approach involves the mutation of editable cytidines to thymidines, with the goal of generating a novel stop codon that ultimately results in a nonfunctional gene product. The workflow involves two major sections, one for the identification of editable cytidines, the design of the targeting spacer oligonucleotides for introduction into the CRISPR-nCas9 cytidine deaminase plasmid, and the construction of the gene-targeting CRISPR-nCas9 cytosine deaminase plasmids, and one for the actual introduction of the mutation in the species of interest. Here, we describe the steps for the first part. To better illustrate the method and oligonucleotide design, we describe the construction of Staphylococcus aureus RN4220 geh mutants with C to T base changes at two different positions, leading to the construction of strains RN4220-geh(160stop) and RN4220-geh(712stop). We outline the steps for (1) the identification of editable cytidines within genes using the CRISPR-CBEI toolkit website, and (2) the design of the targeting spacer oligonucleotides for introduction into the CRISPR-nCas9 cytidine deaminase plasmid pnCasSA-BEC, followed by (3) the construction of the gene-targeting (in this example, geh gene-targeting) CRISPR-nCas9 cytosine deaminase plasmids pnCasSA-BEC-gehC160T and pnCasSA-BEC-gehC712T using the Golden Gate assembly method, plasmid recovery in Escherichia coli, and confirmation by colony PCR and sequencing. The method can be easily adapted to construct gene-inactivation mutants in other S. aureus genes.

Development of a versatile nuclease prime editor with upgraded precision.

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.

Mapping the Complete Photocycle that Powers a Large Stokes Shift Red Fluorescent Protein.

Large Stokes shift (LSS) red fluorescent proteins (RFPs) are highly desirable for bioimaging advances. The RFP mKeima, with coexisting cis- and trans-isomers, holds significance as an archetypal system for LSS emission due to excited-state proton transfer (ESPT), yet the mechanisms remain elusive. We implemented femtosecond stimulated Raman spectroscopy (FSRS) and various time-resolved electronic spectroscopies, aided by quantum calculations, to dissect the cis- and trans-mKeima photocycle from ESPT, isomerization, to ground-state proton transfer in solution. This work manifests the power of FSRS with global analysis to resolve Raman fingerprints of intermediate states. Importantly, the deprotonated trans-isomer governs LSS emission at 620 nm, while the deprotonated cis-isomer's 520 nm emission is weak due to an ultrafast cis-to-trans isomerization. Complementary spectroscopic techniques as a table-top toolset are thus essential to study photochemistry in physiological environments.

A novel copper-sensing two-component system for inducing Dsb gene expression in bacteria.

In nature, bacteria must sense copper and tightly regulate gene expression to evade copper toxicity. Here, we identify a new copper-responsive two-component system named DsbRS in the important human pathogen Pseudomonas aeruginosa; in this system, DsbS is a sensor histidine kinase, and DsbR, its cognate response regulator, directly induces the transcription of genes involved in protein disulfide bond formation (Dsb) (i.e., the dsbDEG operon and dsbB). In the absence of copper, DsbS acts as a phosphatase toward DsbR, thus blocking the transcription of Dsb genes. In the presence of copper, the metal ion directly binds to the sensor domain of DsbS, and the Cys82 residue plays a critical role in this process. The copper-binding behavior appears to inhibit the phosphatase activity of DsbS, leading to the activation of DsbR. The copper resistance of the dsbRS knock-out mutant is restored by the ectopic expression of the dsbDEG operon, which is a DsbRS major target. Strikingly, cognates of the dsbRS-dsbDEG pair are widely distributed across eubacteria. In addition, a DsbR-binding site, which contains the consensus sequence 5'-TTA-N8-TTAA-3', is detected in the promoter region of dsbDEG homologs in these species. These findings suggest that the regulation of Dsb genes by DsbRS represents a novel mechanism by which bacterial cells cope with copper stress.

Live-cell RNA imaging using the CRISPR-dCas13 system with modified sgRNAs appended with fluorescent RNA aptamers.

The development of RNA imaging strategies in live cells is essential to improve our understanding of their role in various cellular functions. We report an efficient RNA imaging method based on the CRISPR-dPspCas13b system with fluorescent RNA aptamers in sgRNA (CasFAS) in live cells. Using modified sgRNA attached to fluorescent RNA aptamers that showed reduced background fluorescence, this approach provides a simple, sensitive way to image and track endogenous RNA with high accuracy and efficiency. In addition, color switching can be easily achieved by changing the fluorogenic dye analogues in living cells through user-friendly washing and restaining operations. CasFAS is compatible with orthogonal fluorescent aptamers, such as Broccoli and Pepper, enabling multiple colors RNA labeling or intracellular RNA-RNA interaction imaging. Finally, the visualization of severe fever with thrombocytopenia syndrome virus (SFTSV) was achieved by CasFAS, which may facilitate further studies on this virus.

Virulence-related regulatory network of Pseudomonas syringae.

Transcription factors (TFs) play important roles in regulating multiple biological processes by binding to promoter regions and regulating the global gene transcription levels. Pseudomonas syringae is a Gram-negative phytopathogenic bacterium harbouring 301 putative TFs in its genome, approximately 50 of which are responsible for virulence-related gene and pathway regulation. Over the past decades, RNA sequencing, chromatin immunoprecipitation sequencing, high-throughput systematic evolution of ligands by exponential enrichment, and other technologies have been applied to identify the functions of master regulators and their interactions in virulence-related pathways. This review summarises the recent advances in the regulatory networks of TFs involved in the type III secretion system (T3SS) and non-T3SS virulence-associated pathways, including motility, biofilm formation, quorum sensing, nucleotide-based secondary messengers, phytotoxins, siderophore production, and oxidative stress. Moreover, this review discusses the future perspectives in terms of TF-mediated pathogenesis mechanisms and provides novel insights that will help combat P. syringae infections based on the regulatory networks of TFs.

Anti-infective therapy using species-specific activators of Staphylococcus aureus ClpP.

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.

Guide RNA engineering enables efficient CRISPR editing with a miniature Syntrophomonas palmitatica Cas12f1 nuclease.

Gene therapy is limited by inefficient delivery of large clustered regularly interspaced short palindromic repeat (CRISPR) effectors, such as Cas9 and Cas12a nucleases. Cas12f nucleases are currently one of the most compact CRISPR genome editors. However, the available toolkit of efficient Cas12f editors is limited. Here, we report the characterization and engineering of a miniature CRISPR-Cas12f system from Syntrophomonas palmitatica (SpaCas12f1, 497 amino acids). We show that CRISPR-SpaCas12f1 cleaves double-stranded DNA (dsDNA) with 5' T-rich PAM specificity and is naturally active for genome editing in bacteria. We identify that CRISPR-SpaCas12f1 trans-activating CRISPR RNA (tracrRNA) harbors a unique head-to-toe hairpin structure, and the natural hairpin structure is a key factor in restricting genome editing by SpaCas12f1 in human cells. Systematical engineering of SpaCas12f1 guide RNA transforms CRISPR-SpaCas12f1 into an efficient genome editor comparable to Francisella novicida CRISPR-Cas12a. Our findings expand the mini CRISPR toolbox, paving the way for therapeutic applications of CRISPR-SpaCas12f1 and engineering compact genome manipulation technologies.

Capsule type defines the capability of Klebsiella pneumoniae in evading Kupffer cell capture in the liver.

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.

CRISPR/Cpf1-Mediated Multiplex and Large-Fragment Gene Editing in Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that causes a variety of infections, including life-threatening diseases. Research on S. aureus is constrained by complex and limited genetic manipulation methods. Here, we report a CRISPR/Cpf1-mediated system, pCpfSA, for rapid and versatile genome editing in S. aureus. In direct comparison with the existing CRISPR/Cas9-mediated genome-editing system, the pCpfSA system exhibits enhanced colony-forming units (CFUs) after editing and an expanded targetable range with comparable editing efficiency. Given the precursor crRNA (pre-crRNA) processing activity of Cpf1, the pCpfSA system also allows multiplex gene editing and large-fragment DNA knockout simply by introducing two crRNAs and the corresponding donor templates, which is difficult to achieve using the CRISPR/Cas9 system, thereby greatly expanding the genome editor toolbox for S. aureus.

Genome Editing in Klebsiella pneumoniae Using CRISPR/Cas9 Technology.

CRISPR/Cas9 systems have been widely adopted for genetic manipulation in diverse biological systems owing to the ease of use and high efficiency. We have recently developed a CRISPR/Cas9-based genome editing system (pCasKP-pSGKP) by coupling a CRISPR/Cas9 system with the lambda Red recombination system as well as a cytidine deaminase-mediated base editing system (pBECKP) in Klebsiella pneumoniae, enabling rapid, scarless, and efficient genetic manipulation in diverse K. pneumoniae strains. In this chapter, we introduce the detailed procedures of using these two tools for genome editing in K. pneumoniae.

Molecular basis for cell-wall recycling regulation by transcriptional repressor MurR in Escherichia coli.

The cell-wall recycling process is important for bacterial survival in nutrient-limited conditions and, in certain cases, is directly involved in antibiotic resistance. In the sophisticated cell-wall recycling process in Escherichia coli, the transcriptional repressor MurR controls the expression of murP and murQ, which are involved in transporting and metabolizing N-acetylmuramic acid (MurNAc), generating N-acetylmuramic acid-6-phosphate (MurNAc-6-P) and N-acetylglucosamine-6-phosphate (GlcNAc-6-P). Here, we report that both MurNAc-6-P and GlcNAc-6-P can bind to MurR and weaken the DNA binding ability of MurR. Structural characterizations of MurR in complex with MurNAc-6-P or GlcNAc-6-P as well as in the apo form revealed the detailed ligand recognition chemistries. Further studies showed that only MurNAc-6-P, but not GlcNAc-6-P, is capable of derepressing the expression of murQP controlled by MurR in cells and clarified the substrate specificity through the identification of key residues responsible for ligand binding in the complex structures. In summary, this study deciphered the molecular mechanism of the cell wall recycling process regulated by MurR in E. coli.