CoCas9 is a compact nuclease from the human microbiome for efficient and precise genome editing.

The expansion of the CRISPR-Cas toolbox is highly needed to accelerate the development of therapies for genetic diseases. Here, through the interrogation of a massively expanded repository of metagenome-assembled genomes, mostly from human microbiomes, we uncover a large variety (n = 17,173) of type II CRISPR-Cas loci. Among these we identify CoCas9, a strongly active and high-fidelity nuclease with reduced molecular size (1004 amino acids) isolated from an uncultivated Collinsella species. CoCas9 is efficiently co-delivered with its sgRNA through adeno associated viral (AAV) vectors, obtaining efficient in vivo editing in the mouse retina. With this study we uncover a collection of previously uncharacterized Cas9 nucleases, including CoCas9, which enriches the genome editing toolbox.

Results of a long-term, prospective study on complications of central venous catheter in pediatric patients with hematologic-oncologic diseases.

BACKGROUND: Central venous catheter (CVC)-related complications remain a significant cause of morbidity in pediatric hematology-oncology. We prospectively surveyed the incidence of CVC-related complications in children with hematologic-oncologic diseases. PROCEDURE: Five-hundred-eighty-one CVCs were inserted in 421 patients from January 2010 to June 2022 (153,731 CVC days observation; follow-up data up to December 31, 2022). RESULTS: Overall, 671 complications were recorded (4.365/1000 CVC days): 49.7% malfunctions (1.88/1000 CVC days, 4.8% of CVC early removals), 23.9% bacteremia (0.90/1000, 15.1%), 19.6% mechanical complications (0.74/1000, 70.2%), 20.1% localized infections (0.76/1000, 17.1%), 0.5% thrombosis (0.02/1000, 33.3%). At multivariate analysis, risk factors for malfunction were Broviac-Hickman type of CVC (hazard ratio [HR] 2.5) or Port-a-cath (HR 3.4) or Proline (HR 4.3), p < .0001; for bacteremia double-lumen CVC (HR 3.2, p < .0001); for mechanical complications age at CVC insertion under median (HR 4.5, p < .0001) and Broviac-Hickman (HR 1.6) or Proline (HR 2.7), p = .01; finally for localized infections Broviac-Hickman (HR 2.9) or Proline (HR 4.4), p = .0001. The 2-year cumulative incidence of premature removal was 23.5%, and risk factors were age at CVC insertion under median (HR 2.4, p < .0001), Broviac-Hickman (HR 2.3) or Proline (HR 4.2), p < .0001. CONCLUSIONS: Premature removal occurs in approximately 20%-25% of long-term CVCs. A surveillance program has a fundamental role in identifying the risk factors for CVC complications and the areas of intervention to improve CVC management.

An optimized SpCas9 high-fidelity variant for direct protein delivery.

Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and potential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fidelity Streptococcus pyogenes Cas9 (SpCas9) variants are less active than the wild-type enzyme and are not compatible with RNP delivery. Building on our previous studies on evoCas9, we developed a high-fidelity SpCas9 variant suitable for RNP delivery. The editing efficacy and precision of the recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D substitution, was compared with the R691A mutant (HiFi Cas9), which is currently the only available high-fidelity Cas9 that can be used as an RNP. The comparative analysis was extended to gene substitution experiments where the two high fidelities were used in combination with a DNA donor template, generating different ratios of non-homologous end joining (NHEJ) versus homology-directed repair (HDR) for precise editing. The analyses revealed a heterogeneous efficacy and precision indicating different targeting capabilities between the two variants throughout the genome. The development of rCas9HF, characterized by an editing profile diverse from the currently used HiFi Cas9 in RNP electroporation, increases the genome editing solutions for the highest precision and efficient applications.

Automated identification of sequence-tailored Cas9 proteins using massive metagenomic data.

The identification of the protospacer adjacent motif (PAM) sequences of Cas9 nucleases is crucial for their exploitation in genome editing. Here we develop a computational pipeline that was used to interrogate a massively expanded dataset of metagenome and virome assemblies for accurate and comprehensive PAM predictions. This procedure allows the identification and isolation of sequence-tailored Cas9 nucleases by using the target sequence as bait. As proof of concept, starting from the disease-causing mutation P23H in the RHO gene, we find, isolate and experimentally validate a Cas9 which uses the mutated sequence as PAM. Our PAM prediction pipeline will be instrumental to generate a Cas9 nuclease repertoire responding to any PAM requirement.

Hao-Fountain syndrome and genital disorders: report of a new possible association.

BACKGROUND: Hao-Fountain syndrome is a neurodevelopmental disorder characterized by global developmental delay, variably impaired intellectual development with significant speech delay and, in some males patients, it has been reported an association with hypogonadism. At present less than 50 cases are reported in literature. CASE PRESENTATION: We report a case of this rare syndrome in a young female with isolated tubal torsion; our patients had different hospitalizations without treatment but during the last episode we decide to perform an abdominal surgical explortion. This is the first case in Literature with a new USP7 mutation. CONCLUSIONS: This case opens new perspective in this rare syndrome and a review approach to isolated tubal torsion. These symptoms should be always well checked.

Weekend Mortality in an Italian Hospital: Immediate versus Delayed Bedside Critical Care Treatment.

BACKGROUND: a number of studies highlighted increased mortality associated with hospital admissions during weekends and holidays, the so-call "weekend effect". In this retrospective study of mortality in an acute care public hospital in Italy between 2009 and 2015, we compared inpatient mortality before and after a major organizational change in 2012. The new model (Model 2) implied that the intensivist was available on call from outside the hospital during nighttime, weekends, and holidays. The previous model (Model 1) ensured the presence of the intensivist coordinating a Medical Emergency Team (MET) inside the hospital 24 h a day, 7 days a week. METHODS: life status at discharge after 9298 and 8223 hospital admissions that occurred during two consecutive periods of 1185 days each (organizational Model 1 and 2), respectively, were classified into "discharged alive", "deceased during nighttime-weekends-holidays" and "deceased during daytime-weekdays". We estimated Relative Risk Ratios (RRR) for the associations between the organizational model and life status at discharge using multinomial logistic regression models adjusted for demographic and case-mix indicators, and timing of admission (nighttime-weekends-holidays vs. daytime-weekdays). RESULTS: there were 802 and 840 deaths under Models 1 and 2, respectively. Total mortality was higher for hospital admissions under Model 2 compared to Model 1. Model 2 was associated with a significantly higher risk of death during nighttime-weekends-holidays (IRR: 1.38, 95% CI 1.20-1.59) compared to daytime-weekdays (RRR: 1.12, 95% CI 0.97-1.31) (p = 0.04). Respiratory diagnoses, in particular, acute and chronic respiratory failure (ICD 9 codes 510-519) were the leading causes of the mortality excess under Model 2. CONCLUSIONS: our data suggest that the immediate availability of an intensivist coordinating a MET 24 h, 7 days a week can result in a better prognosis of in-hospital emergencies compared to delayed consultation.