Home-based fecal calprotectin utilization in a general pediatric gastroenterology clinic.

OBJECTIVE: Remote investigation and monitoring have gained importance in ambulatory practice. A home-based fecal calprotectin (FC) test has been developed where the sample is processed and analyzed at home through a smartphone application. We aimed to assess the use of standard ELISA (sFC) versus home-based (hFC) FC testing in a general pediatric gastroenterology clinic. METHODS: Ambulatory pediatric patients with hFC or sFC performed between August 2019 and November 2020 were included. Data regarding demographics, clinical characteristics, medication use, investigations, and final diagnosis, categorized as inflammatory bowel disease (IBD), functional gastrointestinal (GI) disorders, organic non-IBD (ONI) GI disorders, non-GI disorders, and undetermined after 6 months of investigation, were recorded. RESULTS: A total of 453 FC tests from 453 unique patients were included. Of those, 249 (55%) were hFC. FC levels (median) were higher in children with IBD compared to non-IBD diagnosis (sFC 795 vs. 57 µg/g, hFC 595 vs. 47 µg/g, p < 0.001), and in ONI compared to functional GI disorders (sFC 85 vs. 54 µg/g, p = 0.003, hFC 57 vs. 40 µg/g, p < 0.001). No significant difference was observed between different ONI GI disorders or subtypes of functional disorders. Age did not significantly influence levels. CONCLUSIONS: Overall, hFC and sFC provide similar results in the general pediatric GI ambulatory setting. FC is a sensitive but not disease-specific marker to identify patients with IBD. Values appear to be higher in ONI GI disorders over functional disorders, although cut-off values have yet to be determined.

Exploring the role of an artificial intelligence chatbot on appendicitis management: an experimental study on ChatGPT.

BACKGROUND: Appendicitis is a common surgical condition that requires urgent medical attention. Recent advancements in artificial intelligence and large language processing, such as ChatGPT, have demonstrated potential in supporting healthcare management and scientific research. This study aims to evaluate the accuracy and comprehensiveness of ChatGPT's knowledge on appendicitis management. METHODS: Six questions related to appendicitis management were created by experienced RACS qualified general surgeons to assess ChatGPT's ability to provide accurate information. The criteria of ChatGPT answers' accuracy were compared with current healthcare guidelines for appendicitis and subjective evaluation by two RACS qualified General Surgeons. Additionally, ChatGPT was then asked to provide five high level evidence references to support its responses. RESULTS: ChatGPT provided clinically relevant information on appendicitis management, however, was inconsistent in doing so and often provided superficial information. Further to this, ChatGPT encountered difficulties in generating relevant references, with some being either non-existent or incorrect. CONCLUSION: ChatGPT has the potential to provide timely and comprehensible medical information on appendicitis management to laypersons. However, its issue of inaccuracy in information and production of non-existent or erroneous references presents a challenge for researchers and clinicians who may inadvertently employ such information in their research or healthcare. Therefore, clinicians should exercise caution when using ChatGPT for these purposes.

The promiscuous development of an unconventional Qa1b-restricted T cell population.

MHC-E restricted CD8 T cells show promise in vaccine settings, but their development and specificity remain poorly understood. Here we focus on a CD8 T cell population reactive to a self-peptide (FL9) bound to mouse MHC-E (Qa-1b) that is presented in response to loss of the MHC I processing enzyme ERAAP, termed QFL T cells. We find that mature QFL thymocytes are predominantly CD8αß+CD4-, show signs of agonist selection, and give rise to both CD8αα and CD8αß intraepithelial lymphocytes (IEL), as well as memory phenotype CD8αß T cells. QFL T cells require the MHC I subunit ß-2 microglobulin (ß2m), but do not require Qa1b or classical MHC I for positive selection. However, QFL thymocytes do require Qa1b for agonist selection and full functionality. Our data highlight the relaxed requirements for positive selection of an MHC-E restricted T cell population and suggest a CD8αß+CD4- pathway for development of CD8αα IELs.

Deficiency in Galectin-3, -8, and -9 impairs immunity to chronic Mycobacterium tuberculosis infection but not acute infection with multiple intracellular pathogens.

Macrophages employ an array of pattern recognition receptors to detect and eliminate intracellular pathogens that access the cytosol. The cytosolic carbohydrate sensors Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) recognize damaged pathogen-containing phagosomes, and Gal-3 and Gal-8 are reported to restrict bacterial growth via autophagy in cultured cells. However, the contribution of these galectins to host resistance during bacterial infection in vivo remains unclear. We found that Gal-9 binds directly to Mycobacterium tuberculosis (Mtb) and Salmonella enterica serovar Typhimurium (Stm) and localizes to Mtb in macrophages. To determine the combined contribution of membrane damage-sensing galectins to immunity, we generated Gal-3, -8, and -9 triple knockout (TKO) mice. Mtb infection of primary macrophages from TKO mice resulted in defective autophagic flux but normal bacterial replication. Surprisingly, these mice had no discernable defect in resistance to acute infection with Mtb, Stm or Listeria monocytogenes, and had only modest impairments in bacterial growth restriction and CD4 T cell activation during chronic Mtb infection. Collectively, these findings indicate that while Gal-3, -8, and -9 respond to an array of intracellular pathogens, together these membrane damage-sensing galectins play a limited role in host resistance to bacterial infection.

Inferior mesenteric vein preserving lymphadenectomy in high left segmental colectomy for splenic flexure melanoma: A case report.

INTRODUCTION AND IMPORTANCE: Surgical resection is the mainstay for management of splenic flexure cancers, with the aim of achieving adequate lymphadenectomy. Left-sided bowel resections often require ligation of the inferior mesenteric vein (IMV) for mesocolic dissection or lymphadenectomy which can result in congestive colitis on the anal side of the anastomosis secondary to poor venous outflow. Preserving the IMV may mitigate this risk but is technically difficult and can compromise oncological resection. This case report is a rare example of high left segmental resection of the splenic flexure with preservation of the IMV in a patient with splenic flexure melanoma. CASE PRESENTATION: A non-obstructing lesion was discovered in a 73-year-old male who underwent colonoscopy following a positive faecal occult blood test. Biopsy of the lesion confirmed a melanoma. This patient had a history of cutaneous melanoma which was excised 20 years prior. A laparoscopic high left segmental colectomy was performed, and metastatic melanoma was identified in 3 of 12 regional lymph nodes. The patient recovered with no complications. CLINICAL DISCUSSION: This patient underwent high left segmental colectomy to achieve oncological clearance while resecting minimal bowel and preserving bowel function. The IMV was spared in this surgery to prevent venous congestion. Reports of colitis following left sided colectomy have been described, whereby colitis is thought to result from a mismatch in arterial perfusion and venous drainage following IMV resection. CONCLUSION: This case highlights the potential role of preservation of the inferior mesenteric vein in a rare case of splenic flexure melanoma.

Murine cytomegalovirus downregulates ERAAP and induces an unconventional T cell response to self.

The endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) plays a crucial role in shaping the peptide-major histocompatibility complex (MHC) class I repertoire and maintaining immune surveillance. While murine cytomegalovirus (MCMV) has multiple strategies for manipulating the antigen processing pathway to evade immune responses, the host has also developed ways to counter viral immune evasion. In this study, we find that MCMV modulates ERAAP and induces an interferon γ (IFN-γ)-producing CD8+ T cell effector response that targets uninfected ERAAP-deficient cells. We observe that ERAAP downregulation during infection leads to the presentation of the self-peptide FL9 on non-classical Qa-1b, thereby eliciting Qa-1b-restricted QFL T cells to proliferate in the liver and spleen of infected mice. QFL T cells upregulate effector markers upon MCMV infection and are sufficient to reduce viral load after transfer to immunodeficient mice. Our study highlights the consequences of ERAAP dysfunction during viral infection and provides potential targets for anti-viral therapies.

A hierarchy of cell death pathways confers layered resistance to shigellosis in mice.

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease driven by bacterial colonization of colonic intestinal epithelial cells. Vertebrates have evolved programmed cell death pathways that sense invasive enteric pathogens and eliminate their intracellular niche. Previously we reported that genetic removal of one such pathway, the NAIP-NLRC4 inflammasome, is sufficient to convert mice from resistant to susceptible to oral Shigella flexneri challenge (Mitchell et al., 2020). Here, we investigate the protective role of additional cell death pathways during oral mouse Shigella infection. We find that the Caspase-11 inflammasome, which senses Shigella LPS, restricts Shigella colonization of the intestinal epithelium in the absence of NAIP-NLRC4. However, this protection is limited when Shigella expresses OspC3, an effector that antagonizes Caspase-11 activity. TNFα, a cytokine that activates Caspase-8-dependent apoptosis, also provides potent protection from Shigella colonization of the intestinal epithelium when mice lack both NAIP-NLRC4 and Caspase-11. The combined genetic removal of Caspases-1, -11, and -8 renders mice hyper-susceptible to oral Shigella infection. Our findings uncover a layered hierarchy of cell death pathways that limit the ability of an invasive gastrointestinal pathogen to cause disease.

Binary outcomes of enhancer activity underlie stable random monoallelic expression.

Mitotically stable random monoallelic gene expression (RME) is documented for a small percentage of autosomal genes. We developed an in vivo genetic model to study the role of enhancers in RME using high-resolution single-cell analysis of natural killer (NK) cell receptor gene expression and enhancer deletions in the mouse germline. Enhancers of the RME NK receptor genes were accessible and enriched in H3K27ac on silent and active alleles alike in cells sorted according to allelic expression status, suggesting enhancer activation and gene expression status can be decoupled. In genes with multiple enhancers, enhancer deletion reduced gene expression frequency, in one instance converting the universally expressed gene encoding NKG2D into an RME gene, recapitulating all aspects of natural RME including mitotic stability of both the active and silent states. The results support the binary model of enhancer action, and suggest that RME is a consequence of general properties of gene regulation by enhancers rather than an RME-specific epigenetic program. Therefore, many and perhaps all genes may be subject to some degree of RME. Surprisingly, this was borne out by analysis of several genes that define different major hematopoietic lineages, that were previously thought to be universally expressed within those lineages: the genes encoding NKG2D, CD45, CD8α, and Thy-1. We propose that intrinsically probabilistic gene allele regulation is a general property of enhancer-controlled gene expression, with previously documented RME representing an extreme on a broad continuum.

The role of cytokeratin 7/20 coordination revisited-Machine learning identifies improved interpretative algorithms for cell block immunohistochemistry in aspirates of metastatic carcinoma.

BACKGROUND: Fine-needle aspiration (FNA) is a robust diagnostic technique often used for tissue diagnosis of metastatic carcinoma. For interpretation of FNA cytology, cell block immunohistochemistry (IHC) and clinicocytologic parameters are indispensable. In this review of a large cohort, the current report: 1) describes clinicocytologic parameters and immunoprofiles of aspirates of metastatic carcinoma, 2) compares the predictivity of immunostains and classical approaches for IHC interpretation, and 3) describes machine learning-based algorithms for IHC interpretation. METHODS: Aspirates of metastatic carcinoma that had IHC performed were retrieved. Clinicocytologic parameters, IHC results, the corresponding primary site, and histologic diagnoses were recorded. By using machine learning, decision trees for predicting the primary site were generated, their performance was compared with 2 human-designed algorithms, and the primary site was suggested in the historical diagnosis. RESULTS: In total, 1145 cases were identified. The 6 most populated groups were selected for machine learning and predictive analysis. With IHC input, the decision tree achieved a concordance rate of 94.5% and overall accuracy of 83.6%, which improved to 95.3% and 85.8%, respectively, when clinical data were incorporated and exceeded the human-designed IHC algorithms (P < .001). The historical diagnosis was more accurate unless indeterminate diagnoses were regarded as discordant (P < .001). CDX2 and TTF-1 immunostains had the highest weight in model accuracy, occupied the root of the decision trees, scored higher as features of importance, and outperformed the predictive power of cytokeratins 7 and 20. CONCLUSIONS: Cytokeratins 7 and 20 may be superseded in immunostaining panels, including organ-specific immunostains such as CDX2 and TTF-1. Machine learning generates algorithms that surpasses human-designed algorithms but is inferior to expert assessment integrating clinical and cytologic assessment.

A mouse-specific retrotransposon drives a conserved Cdk2ap1 isoform essential for development.

Retrotransposons mediate gene regulation in important developmental and pathological processes. Here, we characterized the transient retrotransposon induction during preimplantation development of eight mammals. Induced retrotransposons exhibit similar preimplantation profiles across species, conferring gene regulatory activities, particularly through long terminal repeat (LTR) retrotransposon promoters. A mouse-specific MT2B2 retrotransposon promoter generates an N-terminally truncated Cdk2ap1ΔN that peaks in preimplantation embryos and promotes proliferation. In contrast, the canonical Cdk2ap1 peaks in mid-gestation and represses cell proliferation. This MT2B2 promoter, whose deletion abolishes Cdk2ap1ΔN production, reduces cell proliferation and impairs embryo implantation, is developmentally essential. Intriguingly, Cdk2ap1ΔN is evolutionarily conserved in sequence and function yet is driven by different promoters across mammals. The distinct preimplantation Cdk2ap1ΔN expression in each mammalian species correlates with the duration of its preimplantation development. Hence, species-specific transposon promoters can yield evolutionarily conserved, alternative protein isoforms, bestowing them with new functions and species-specific expression to govern essential biological divergence.

Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons.

Type I interferons (IFNs) are essential for anti-viral immunity, but often impair protective immune responses during bacterial infections. An important question is how type I IFNs are strongly induced during viral infections, and yet are appropriately restrained during bacterial infections. The Super susceptibility to tuberculosis 1 (Sst1) locus in mice confers resistance to diverse bacterial infections. Here we provide evidence that Sp140 is a gene encoded within the Sst1 locus that represses type I IFN transcription during bacterial infections. We generated Sp140-/- mice and found that they are susceptible to infection by Legionella pneumophila and Mycobacterium tuberculosis. Susceptibility of Sp140-/- mice to bacterial infection was rescued by crosses to mice lacking the type I IFN receptor (Ifnar-/-). Our results implicate Sp140 as an important negative regulator of type I IFNs that is essential for resistance to bacterial infections.

Generation of a DAT-P2A-Flpo mouse line for intersectional genetic targeting of dopamine neuron subpopulations.

Dopaminergic projections exert widespread influence over multiple brain regions and modulate various behaviors including movement, reward learning, and motivation. It is increasingly appreciated that dopamine neurons are heterogeneous in their gene expression, circuitry, physiology, and function. Current approaches to target dopamine neurons are largely based on single gene drivers, which either label all dopamine neurons or mark a subset but concurrently label non-dopaminergic neurons. Here, we establish a mouse line with Flpo recombinase expressed from the endogenous Slc6a3 (dopamine active transporter [DAT]) locus. DAT-P2A-Flpo mice can be used together with Cre-expressing mouse lines to efficiently and selectively label dopaminergic subpopulations using Cre/Flp-dependent intersectional strategies. We demonstrate the utility of this approach by generating DAT-P2A-Flpo;NEX-Cre mice that specifically label Neurod6-expressing dopamine neurons, which project to the nucleus accumbens medial shell. DAT-P2A-Flpo mice add to a growing toolbox of genetic resources that will help parse the diverse functions mediated by dopaminergic circuits.

Deep learning model for prediction of extended-spectrum beta-lactamase (ESBL) production in community-onset Enterobacteriaceae bacteraemia from a high ESBL prevalence multi-centre cohort.

Adequate empirical antimicrobial coverage is instrumental in clinical management of community-onset Enterobacteriaceae bacteraemia in areas with high ESBL prevalence, while balancing the risk of carbapenem overuse and emergence of carbapenem-resistant organisms. It is unknown whether machine learning offers additional advantages to conventional statistical methods in prediction of ESBL production. To develop a validated model to predict ESBL production in Enterobacteriaceae causing community-onset bacteraemia. 5625 patients with community-onset bacteraemia caused by Escherichia coli, Klebsiella species and Proteus mirabilis during 1 January 2015-31 December 2019 from three regional hospitals in Hong Kong were included in the analysis, after exclusion of blood cultures obtained beyond 48 h of admission. The prevalence of ESBL-producing Enterobacteriaceae was 23.7% (1335/5625). Deep neural network and other machine learning algorithms were compared against conventional statistical model via multivariable logistic regression. Primary outcomes compared consisted of predictive model area under curve of receiver-operator characteristic curve (AUC), and macro-averaged F1 score. Secondary outcomes included sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Deep neural network yielded an AUC of 0.761 (95% CI 0.725-0.797) and F1 score of 0.661 (95% CI 0.633-0.689), which was superior to logistic regression (AUC 0.667 (95% CI 0.627-0.707), F1 score 0.596 (95% CI 0.567-0.625)). Deep neural network had a specificity of 91.5%, sensitivity of 37.5%, NPV of 82.5%, and PPV of 57.9%. Deep neural network is superior to logistic regression in predicting ESBL production in Enterobacteriaceae causing community-onset bacteraemia in high-ESBL prevalence area. Machine learning offers clinical utility in guiding judicious empirical antibiotics use.

Male-biased fasting-induced changes in hepatic tauro-cholic acid and plasma cholesterol in Sult2a8-haplodeficient mice.

SULT2A8 is a male-predominant and liver-specific mouse cytosolic sulfotransferase (SULT) that sulfonates 7α-hydroxyl (7α-OH) bile acids in vitro. Sulfonation regulates bile acid homeostasis, which in turn regulates cholesterol and energy metabolism. Using the Sult2a8-heterozygous (HT) mouse model created earlier in our laboratory, we aimed to investigate the physiological role of SULT2A8 in sulfonating 7α-OH bile acids and its impact on energy metabolism in vivo under both fed and energy-deprivation conditions. Disruption of one allele of the Sult2a8 gene in male HT mice resulted in losing ~ 50% of the 7α-OH sulfonating activity compared to wild-type (WT) control, but no significant change in female HT mice. Under the fed condition comparing the levels of hepatic and biliary bile acids as well as plasma/serum energy metabolites, HT mice displayed a profile similar to that of WT mice, suggesting that the Sult2a8-haplodeficient mice conducted normal energy metabolism. However, after 48-h fasting, a significant decrease in plasma cholesterol level was found in male HT mice but without any significant reduction in female HT mice. Of interest, in male Sult2a8-haplodeficient mice, an increase of the hepatic taurine-conjugated cholic acid level was noted but no noticeable change in other tested bile acids after fasting. Taken together, SULT2A8 is a male-specific and key hepatic SULT in metabolizing 7α-OH primary bile acids. During energy deprivation, SULT2A8 is required to maintain the bile acid and cholesterol metabolism, suggesting SULT is a potential therapeutic target for controlling metabolic diseases.

NAIP-NLRC4-deficient mice are susceptible to shigellosis.

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease that is a major cause of diarrhea-associated mortality in humans. Mice are highly resistant to Shigella and the lack of a tractable physiological model of shigellosis has impeded our understanding of this important human disease. Here, we propose that the differential susceptibility of mice and humans to Shigella is due to mouse-specific activation of the NAIP-NLRC4 inflammasome. We find that NAIP-NLRC4-deficient mice are highly susceptible to oral Shigella infection and recapitulate the clinical features of human shigellosis. Although inflammasomes are generally thought to promote Shigella pathogenesis, we instead demonstrate that intestinal epithelial cell (IEC)-specific NAIP-NLRC4 activity is sufficient to protect mice from shigellosis. In addition to describing a new mouse model of shigellosis, our results suggest that the lack of an inflammasome response in IECs may help explain the susceptibility of humans to shigellosis.

Interferon-independent STING signaling promotes resistance to HSV-1 in vivo.

The Stimulator of Interferon Genes (STING) pathway initiates potent immune responses upon recognition of DNA. To initiate signaling, serine 365 (S365) in the C-terminal tail (CTT) of STING is phosphorylated, leading to induction of type I interferons (IFNs). Additionally, evolutionary conserved responses such as autophagy also occur downstream of STING, but their relative importance during in vivo infections remains unclear. Here we report that mice harboring a serine 365-to-alanine (S365A) mutation in STING are unexpectedly resistant to Herpes Simplex Virus (HSV)-1, despite lacking STING-induced type I IFN responses. By contrast, resistance to HSV-1 is abolished in mice lacking the STING CTT, suggesting that the STING CTT initiates protective responses against HSV-1, independently of type I IFNs. Interestingly, we find that STING-induced autophagy is a CTT- and TBK1-dependent but IRF3-independent process that is conserved in the STING S365A mice. Thus, interferon-independent functions of STING mediate STING-dependent antiviral responses in vivo.

Aifm2, a NADH Oxidase, Supports Robust Glycolysis and Is Required for Cold- and Diet-Induced Thermogenesis.

Brown adipose tissue (BAT) is highly metabolically active tissue that dissipates energy via UCP1 as heat, and BAT mass is correlated negatively with obesity. The presence of BAT/BAT-like tissue in humans renders BAT as an attractive target against obesity and insulin resistance. Here, we identify Aifm2, a NADH oxidoreductase domain containing flavoprotein, as a lipid droplet (LD)-associated protein highly enriched in BAT. Aifm2 is induced by cold as well as by diet. Upon cold or ß-adrenergic stimulation, Aifm2 associates with the outer side of the mitochondrial inner membrane. As a unique BAT-specific first mammalian NDE (external NADH dehydrogenase)-like enzyme, Aifm2 oxidizes NADH to maintain high cytosolic NAD levels in supporting robust glycolysis and to transfer electrons to the electron transport chain (ETC) for fueling thermogenesis. Aifm2 in BAT and subcutaneous white adipose tissue (WAT) promotes oxygen consumption, uncoupled respiration, and heat production during cold- and diet-induced thermogenesis. Aifm2, thus, can ameliorate diet-induced obesity and insulin resistance.

IRG1 and Inducible Nitric Oxide Synthase Act Redundantly with Other Interferon-Gamma-Induced Factors To Restrict Intracellular Replication of Legionella pneumophila.

Interferon gamma (IFN-γ) restricts the intracellular replication of many pathogens, but the mechanism by which IFN-γ confers cell-intrinsic pathogen resistance remains unclear. For example, intracellular replication of the bacterial pathogen Legionella pneumophila in macrophages is potently curtailed by IFN-γ. However, consistent with prior studies, no individual genetic deficiency that we tested completely abolished IFN-γ-mediated control. Intriguingly, we observed that the glycolysis inhibitor 2-deoxyglucose (2DG) partially rescued L. pneumophila replication in IFN-γ-treated macrophages. 2DG inhibits glycolysis and triggers the unfolded protein response, but unexpectedly, it appears these effects are not responsible for perturbing the antimicrobial activity of IFN-γ. Instead, we found that 2DG rescues bacterial replication by inhibiting the expression of two key antimicrobial factors, inducible nitric oxide synthase (iNOS) and immune-responsive gene 1 (IRG1). Using immortalized and primary macrophages deficient in iNOS and IRG1, we confirmed that loss of both iNOS and IRG1, but not individual deficiency in either gene, partially reduced IFN-γ-mediated restriction of L. pneumophila Further, using a combinatorial CRISPR/Cas9 mutagenesis approach, we found that mutation of iNOS and IRG1 in combination with four other genes (CASP11, IRGM1, IRGM3, and NOX2) resulted in a total loss of L. pneumophila restriction by IFN-γ in primary bone marrow macrophages. Our study defines a complete set of cell-intrinsic factors required for IFN-γ-mediated restriction of an intracellular bacterial pathogen and highlights the combinatorial strategy used by hosts to block bacterial replication in macrophages.IMPORTANCELegionella pneumophila is one example among many species of pathogenic bacteria that replicate within mammalian macrophages during infection. The immune signaling factor interferon gamma (IFN-γ) blocks L. pneumophila replication in macrophages and is an essential component of the immune response to L. pneumophila and other intracellular pathogens. However, to date, no study has identified the exact molecular factors induced by IFN-γ that are required for its activity. We generated macrophages lacking different combinations of IFN-γ-induced genes in an attempt to find a genetic background in which there is a complete loss of IFN-γ-mediated restriction of L. pneumophila We identified six genes that comprise the totality of the IFN-γ-dependent restriction of L. pneumophila replication in macrophages. Our results clarify the molecular basis underlying the potent effects of IFN-γ and highlight how redundancy downstream of IFN-γ is key to prevent exploitation of macrophages by pathogens.

CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection.

Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging, conditional knockout, site-specific transgene insertion, and gene replacement; however, existing methods to generate such animals remain laborious and costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP electroporation and AAV donor infection), combining adeno-associated virus (AAV)-mediated HDR donor delivery with Cas9/sgRNA RNP electroporation to engineer large site-specific modifications in the mouse genome with high efficiency and throughput. We successfully targeted a 774 bp fluorescent reporter, a 2.1 kb CreERT2 driver, and a 3.3 kb expression cassette into endogenous loci in both embryos and live mice. CRISPR-READI is applicable to most widely used knockin schemes requiring donor lengths within the 4.9 kb AAV packaging capacity. Altogether, CRISPR-READI is an efficient, high-throughput, microinjection-free approach for sophisticated mouse genome engineering with potential applications in other mammalian species.

Qa-1-Restricted CD8+ T Cells Can Compensate for the Absence of Conventional T Cells during Viral Infection.

The role of non-classical T cells during viral infection remains poorly understood. Using the well-established murine model of CMV infection (MCMV) and mice deficient in MHC class Ia molecules, we found that non-classical CD8+ T cells robustly expand after MCMV challenge, become highly activated effectors, and are capable of forming durable memory. Interestingly, although these cells are restricted by MHC class Ib molecules, they respond similarly to conventional T cells. Remarkably, when acting as the sole component of the adaptive immune response, non-classical CD8+ T cells are sufficient to protect against MCMV-induced lethality. We also demonstrate that the MHC class Ib molecule Qa-1 (encoded by H2-T23) restricts a large, and critical, portion of this population. These findings reveal a potential adaptation of the host immune response to compensate for viral evasion of classical T cell immunity.