Continuous directed evolution of a compact CjCas9 variant with broad PAM compatibility

CRISPR-Cas9 genome engineering is a powerful technology for correcting genetic diseases. However, the targeting range of Cas9 proteins is limited by their requirement for a protospacer adjacent motif (PAM), and in vivo delivery is challenging due to their large size. Here, we use phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 ortholog characterized to date. The identified variant, termed evoCjCas9, primarily recognizes N$_{4}$AH and N$_{5}$HA PAM sequences, which occur tenfold more frequently in the genome than the canonical N$_{3}$VRYAC PAM site. Moreover, evoCjCas9 exhibits higher nuclease activity than wild-type CjCas9 on canonical PAMs, with editing rates comparable to commonly used PAM-relaxed SpCas9 variants. Combined with deaminases or reverse transcriptases, evoCjCas9 enables robust base and prime editing, with the small size of evoCjCas9 base editors allowing for tissue-specific installation of A-to-G or C-to-T transition mutations from single adeno-associated virus vector systems

Treatment of a metabolic liver disease by in vivo prime editing in mice

Prime editing is a highly versatile CRISPR-based genome editing technology with the potential to correct the vast majority of pathogenic mutations (1). However, correction of a disease phenotype in vivo in somatic tissues has not been demonstrated thus far. Here, we establish proof-of-concept for in vivo prime editing and repair the metabolic liver disease phenylketonuria (PKU) in mice. We first developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain of the reverse transcriptase (PE2ΔRnH), and a linker- and NLS-optimized intein-split PE construct (PE2 p.1153) for delivery by adeno-associated virus (AAV) vectors. Systemic dual AAV-mediated delivery of this variant into the liver of neonatal mice enabled installation of a transversion mutation at the Dnmt1 locus with an average efficiency of 15%, and delivery of unsplit PE2ΔRnH using human adenoviral vector 5 (AdV5) further increased editing rates to 58%. PE2ΔRnH-encoding AdV5 was also used to correct the disease-causing mutation of the phenylalanine hydroxylase (Pah)enu2 allele in phenylketonuria (PKU) mice with an average efficiency of 8% (up to 17.3%), leading to therapeutic reduction of blood phenylalanine (L-Phe) levels. Our study demonstrates in vivo prime editing in the liver with high precision and editing rates sufficient to treat a number of metabolic liver diseases, emphasizing the potential of prime editing for future therapeutic applications

In vivo prime editing of a metabolic liver disease in mice

Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain (PE2$^{ΔRnH}$) and an intein-split construct (PE2 p.1153) for adeno-associated virus–mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2$^{ΔRnH}$ via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pah $^{enu2}$ mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 10 $^{14}$ vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases

In vivo prime editing of a metabolic liver disease in mice

Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain (PE2ΔRnH) and an intein-split construct (PE2 p.1153) for adeno-associated virus-mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2ΔRnH via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pahenu2 mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 1014 vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.ISSN:1946-6234ISSN:1946-624

The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila

The amoeba-resistant bacterium Legionella pneumophila causes Legionnaires' disease and employs a type IV secretion system (T4SS) to replicate in the unique, ER-associated Legionella-containing vacuole (LCV). The large fusion GTPase Sey1/atlastin is implicated in ER dynamics, ER-derived lipid droplet (LD) formation, and LCV maturation. Here, we employ cryo-electron tomography, confocal microscopy, proteomics, and isotopologue profiling to analyze LCV-LD interactions in the genetically tractable amoeba Dictyostelium discoideum. Dually fluorescence-labeled D. discoideum producing LCV and LD markers revealed that Sey1 as well as the L. pneumophila T4SS and the Ran GTPase activator LegG1 promote LCV-LD interactions. In vitro reconstitution using purified LCVs and LDs from parental or Delta sey1 mutant D. discoideum indicated that Sey1 and GTP promote this process. Sey1 and the L. pneumophila fatty acid transporter FadL were implicated in palmitate catabolism and palmitate-dependent intracellular growth. Taken together, our results reveal that Sey1 and LegG1 mediate LD- and FadL-dependent fatty acid metabolism of intracellular L. pneumophila.ISSN:2050-084

Salmonella Typhimurium discreet-invasion of the murine gut absorptive epithelium.

Salmonella enterica serovar Typhimurium (S.Tm) infections of cultured cell lines have given rise to the ruffle model for epithelial cell invasion. According to this model, the Type-Three-Secretion-System-1 (TTSS-1) effectors SopB, SopE and SopE2 drive an explosive actin nucleation cascade, resulting in large lamellipodia- and filopodia-containing ruffles and cooperative S.Tm uptake. However, cell line experiments poorly recapitulate many of the cell and tissue features encountered in the hosts gut mucosa. Here, we employed bacterial genetics and multiple imaging modalities to compare S.Tm invasion of cultured epithelial cell lines and the gut absorptive epithelium in vivo in mice. In contrast to the prevailing ruffle-model, we find that absorptive epithelial cell entry in the mouse gut occurs through discreet-invasion. This distinct entry mode requires the conserved TTSS-1 effector SipA, involves modest elongation of local microvilli in the absence of expansive ruffles, and does not favor cooperative invasion. Discreet-invasion preferentially targets apicolateral hot spots at cell-cell junctions and shows strong dependence on local cell neighborhood. This proof-of-principle evidence challenges the current model for how S.Tm can enter gut absorptive epithelial cells in their intact in vivo context

Enhancing prime editor activity by directed protein evolution in yeast

Prime editing is a highly versatile genome editing technology that enables the introduction of base substitutions, insertions, and deletions. However, compared to traditional Cas9 nucleases prime editors (PEs) are less active. In this study we use OrthoRep, a yeast-based platform for directed protein evolution, to enhance the editing efficiency of PEs. After several rounds of evolution with increased selection pressure, we identify multiple mutations that have a positive effect on PE activity in yeast cells and in biochemical assays. Combining the two most effective mutations – the A259D amino acid substitution in nCas9 and the K445T substitution in M-MLV RT – results in the variant PE_Y18. Delivery of PE_Y18, encoded on DNA, mRNA or as a ribonucleoprotein complex into mammalian cell lines increases editing rates up to 3.5-fold compared to PEmax. In addition, PE_Y18 supports higher prime editing rates when delivered in vivo into the liver or brain. Our study demonstrates proof-of-concept for the application of OrthoRep to optimize genome editing tools in eukaryotic cells.ISSN:2041-172

Intestinal epithelial NAIP/NLRC4 restricts systemic dissemination of the adapted pathogen Salmonella Typhimurium due to site-specific bacterial PAMP expression.

Inflammasomes can prevent systemic dissemination of enteropathogenic bacteria. As adapted pathogens including Salmonella Typhimurium (S. Tm) have evolved evasion strategies, it has remained unclear when and where inflammasomes restrict their dissemination. Bacterial population dynamics establish that the NAIP/NLRC4 inflammasome specifically restricts S. Tm migration from the gut to draining lymph nodes. This is solely attributable to NAIP/NLRC4 within intestinal epithelial cells (IECs), while S. Tm evades restriction by phagocyte NAIP/NLRC4. NLRP3 and Caspase-11 also fail to restrict S. Tm mucosa traversal, migration to lymph nodes, and systemic pathogen growth. The ability of IECs (not phagocytes) to mount a NAIP/NLRC4 defense in vivo is explained by particularly high NAIP/NLRC4 expression in IECs and the necessity for epithelium-invading S. Tm to express the NAIP1-6 ligands-flagella and type-III-secretion-system-1. Imaging reveals both ligands to be promptly downregulated following IEC-traversal. These results highlight the importance of intestinal epithelial NAIP/NLRC4 in blocking bacterial dissemination in vivo, and explain why this constitutes a uniquely evasion-proof defense against the adapted enteropathogen S. Tm

Salmonella Typhimurium discreet-invasion of the murine gut absorptive epithelium

Salmonella enterica serovar Typhimurium (S.Tm) infections of cultured cell lines have given rise to the ruffle model for epithelial cell invasion. According to this model, the Type-Three-Secretion-System-1 (TTSS-1) effectors SopB, SopE and SopE2 drive an explosive actin nucleation cascade, resulting in large lamellipodia- and filopodia-containing ruffles and cooperative S.Tm uptake. However, cell line experiments poorly recapitulate many of the cell and tissue features encountered in the host’s gut mucosa. Here, we employed bacterial genetics and multiple imaging modalities to compare S.Tm invasion of cultured epithelial cell lines and the gut absorptive epithelium in vivo in mice. In contrast to the prevailing ruffle-model, we find that absorptive epithelial cell entry in the mouse gut occurs through “discreet-invasion”. This distinct entry mode requires the conserved TTSS-1 effector SipA, involves modest elongation of local microvilli in the absence of expansive ruffles, and does not favor cooperative invasion. Discreet-invasion preferentially targets apicolateral hot spots at cell–cell junctions and shows strong dependence on local cell neighborhood. This proof-of-principle evidence challenges the current model for how S.Tm can enter gut absorptive epithelial cells in their intact in vivo context.ISSN:1553-7374ISSN:1553-736

Intestinal epithelial NAIP/NLRC4 restricts systemic dissemination of the adapted pathogen Salmonella Typhimurium due to site-specific bacterial PAMP expression

Inflammasomes can prevent systemic dissemination of enteropathogenic bacteria. As adapted pathogens including Salmonella Typhimurium (S. Tm) have evolved evasion strategies, it has remained unclear when and where inflammasomes restrict their dissemination. Bacterial population dynamics establish that the NAIP/NLRC4 inflammasome specifically restricts S. Tm migration from the gut to draining lymph nodes. This is solely attributable to NAIP/NLRC4 within intestinal epithelial cells (IECs), while S. Tm evades restriction by phagocyte NAIP/NLRC4. NLRP3 and Caspase-11 also fail to restrict S. Tm mucosa traversal, migration to lymph nodes, and systemic pathogen growth. The ability of IECs (not phagocytes) to mount a NAIP/NLRC4 defense in vivo is explained by particularly high NAIP/NLRC4 expression in IECs and the necessity for epithelium-invading S. Tm to express the NAIP1-6 ligands—flagella and type-III-secretion-system-1. Imaging reveals both ligands to be promptly downregulated following IEC-traversal. These results highlight the importance of intestinal epithelial NAIP/NLRC4 in blocking bacterial dissemination in vivo, and explain why this constitutes a uniquely evasion-proof defense against the adapted enteropathogen S. Tm