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Prime editing enables search-and-replace genome editing but is limited by low editing efficiency. We present a high-throughput approach, the Peptide Self-Editing sequencing assay (PepSEq), to measure how fusion of 12,000 85-amino acid peptides influences prime editing efficiency. We show that peptide fusion can enhance prime editing, prime-enhancing peptides combine productively, and a top dual peptide-prime editor increases prime editing significantly in multiple cell lines across dozens of target sites. Top prime-enhancing peptides function by increasing translation efficiency and serve as broadly useful tools to improve prime editing efficiency. Prime editing enables search-and-replace genome editing but is limited by low editing efficiency. Here the authors present PepSEq, a high-throughput method for screening a large library of peptides that influence prime editing efficiency.

The small intestine is a rapidly proliferating organ that is maintained by a small population of Lgr5 -expressing intestinal stem cells (ISCs). However, several Lgr5 -negative ISC populations have been identified, and this remarkable plasticity allows the intestine to rapidly respond to both the local environment and to damage. However, the mediators of such plasticity are still largely unknown. Using intestinal organoids and mouse models, we show that upon ribosome impairment (driven by Rptor deletion, amino acid starvation, or low dose cyclohexamide treatment) ISCs gain an Lgr5 -negative, fetal-like identity. This is accompanied by a rewiring of metabolism. Our findings suggest that the ribosome can act as a sensor of nutrient availability, allowing ISCs to respond to the local nutrient environment. Mechanistically, we show that this phenotype requires the activation of ZAKɑ, which in turn activates YAP, via SRC. Together, our data reveals a central role for ribosome dynamics in intestinal stem cells, and identify the activation of ZAKɑ as a critical mediator of stem cell identity. Intestinal stem cells are responsible for replenishing cells within the high-turnover intestinal epithelium. Here they show that ribosome dynamics affect intestinal stem cell identity through a mechanism that is triggered by changes in nutrient availability.

Genomics for rare disease diagnosis has advanced at a rapid pace due to our ability to perform in-depth analyses on individual patients with ultra-rare diseases. The increasing sizes of ultra-rare disease cohorts internationally newly enables cohort-wide analyses for new discoveries, but well-calibrated statistical genetics approaches for jointly analyzing these patients are still under development. The Undiagnosed Diseases Network (UDN) brings multiple clinical, research and experimental centers under the same umbrella across the United States to facilitate and scale case-based diagnostic analyses. Here, we present the first joint analysis of whole genome sequencing data of UDN patients across the network. We introduce new, well-calibrated statistical methods for prioritizing disease genes with de novo recurrence and compound heterozygosity. We also detect pathways enriched with candidate and known diagnostic genes. Our computational analysis, coupled with a systematic clinical review, recapitulated known diagnoses and revealed new disease associations. We further release a software package, RaMeDiES, enabling automated cross-analysis of deidentified sequenced cohorts for new diagnostic and research discoveries. Gene-level findings and variant-level information across the cohort are available in a public-facing browser ( https://dbmi-bgm.github.io/udn-browser/ ). These results show that case-level diagnostic efforts should be supplemented by a joint genomic analysis across cohorts. Using well-calibrated statistical methods the authors jointly analyze Undiagnosed Diseases Network genomes, identifying known and novel disease genes. Software is publicly available to support future cross-cohort rare disease discovery efforts.

Genomics for rare disease diagnosis has advanced at a rapid pace due to our ability to perform \"N-of-1\" analyses on individual patients with ultra-rare diseases. The increasing sizes of ultra-rare disease cohorts internationally newly enables cohort-wide analyses for new discoveries, but well-calibrated statistical genetics approaches for jointly analyzing these patients are still under development. The Undiagnosed Diseases Network (UDN) brings multiple clinical, research and experimental centers under the same umbrella across the United States to facilitate and scale N-of-1 analyses. Here, we present the first joint analysis of whole genome sequencing data of UDN patients across the network. We introduce new, well-calibrated statistical methods for prioritizing disease genes with recurrence and compound heterozygosity. We also detect pathways enriched with candidate and known diagnostic genes. Our computational analysis, coupled with a systematic clinical review, recapitulated known diagnoses and revealed new disease associations. We further release a software package, RaMeDiES, enabling automated cross-analysis of deidentified sequenced cohorts for new diagnostic and research discoveries. Gene-level findings and variant-level information across the cohort are available in a public-facing browser (https://dbmi-bgm.github.io/udn-browser/). These results show that N-of-1 efforts should be supplemented by a joint genomic analysis across cohorts.

Prime editing enables search-and-replace genome editing but is limited by low editing efficiency. We present a high-throughput approach, PepSEq, to measure how fusion of 12,000 85-amino acid peptides derived from human DNA repair-related proteins influences prime editing efficiency. We show that peptide fusion can enhance prime editing, prime-enhancing peptides combine productively, and a top dual peptide-prime editor increases prime editing significantly in multiple cell lines across dozens of target sites. Competing Interest Statement The authors have declared no competing interest.

The purpose of this study was to explore sampling options for a reliable and logistically more feasible protocol during a large EHV-1 outbreak. Seventeen horses with clinical infection as well as nineteen healthy herdmates, all part of an EHM outbreak, were enrolled in the study. Each horse was sampled two–four times at intervals of 2–6 days during the outbreak. All samples were collected using 6′′ rayon-tipped swabs. Nasal secretions were used as the diagnostic sample of choice. Additional samples, including swabs from the muzzle/nares, swabs from the front limbs, rectal swabs, swabs of the feed bin, and swabs of the water troughs were collected as well. All swabs were tested for the presence of EHV-1 by qPCR. With the exception of two EHV-1 qPCR-positive swabs from two different horses, all remaining swabs collected from healthy herdmates tested qPCR-negative for EHV-1. For horses with clinical infection, EHV-1 was detected in 31 nasal swabs, 30 muzzle/nares swabs, 7 front limb swabs, 7 feeders, 6 water troughs and 6 rectal swabs. Not all positive muzzle/nares swabs correlated with a positive nasal swab from the same set, however, and all other positive swabs did correlate with a positive nasal swab in their respective set. The agreement between nasal swabs and muzzle/nares swabs was 74%. The sampling of non-invasive swabs from the muzzle/nares should facilitate the identification of EHV-1 shedders during an outbreak, allowing for prompt isolation and implementation of biosecurity measures.

Neutralizing antibody responses gradually wane after vaccination with mRNA-1273 against several variants of concern (VOC), and additional boost vaccinations may be required to sustain immunity and protection. Here, we evaluated the immune responses in nonhuman primates that received 100 μg of mRNA-1273 vaccine at 0 and 4 weeks and were boosted at week 29 with mRNA-1273 (homologous) or mRNA-1273.β (heterologous), which encompasses the spike sequence of the B.1.351 (beta or β) variant. Reciprocal ID50 pseudovirus neutralizing antibody geometric mean titers (GMT) against live SARS-CoV-2 D614G and the β variant, were 4700 and 765, respectively, at week 6, the peak of primary response, and 644 and 553, respectively, at a 5-month post-vaccination memory time point. Two weeks following homologous or heterologous boost β-specific reciprocal ID50 GMT were 5000 and 3000, respectively. At week 38, animals were challenged in the upper and lower airway with the β variant. Two days post-challenge, viral replication was low to undetectable in both BAL and nasal swabs in most of the boosted animals. These data show that boosting with the homologous mRNA-1273 vaccine six months after primary immunization provides up to a 20-fold increase in neutralizing antibody responses across all VOC, which may be required to sustain high-level protection against severe disease, especially for at-risk populations. Competing Interest Statement K.S.C. and B.S.G. are inventors on U.S. Patent No. 10,960,070 B2 and International Patent Application No. WO/2018/081318 entitled Prefusion Coronavirus Spike Proteins and Their Use. K.S.C. and B.S.G. are inventors on US Patent Application No. 62/972,886 entitled 2019-nCoV Vaccine. J.M., L.W., C.A.S., J.R.M, D.D, N.J.S., A.R.H., W.S., Y.Z. and M.R. are inventors on US patent application No. 63/147,419 entitled Antibodies Targeting the Spike Protein of Coronaviruses. D.V., A.V.R., Z.F., A.C., L.P., H.A., and M.G.L. are employees of Bioqual. K.S.C, B.S.G, L.W., W.S., and Y.Z. are inventors on multiple US Patent Applications entitled Anti-Coronavirus Antibodies and Methods of Use. A.C., M.K., A.C., and D.K.E. are employees of Moderna. M.S.S. serves on the scientific board of advisors for Moderna.

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. We immunized rhesus macaques at weeks 0 and 4 and assessed immune responses over one year in blood, upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID 50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody binding titers also decreased in bronchoalveolar lavage (BAL). Four days after challenge, virus was unculturable in BAL and subgenomic RNA declined ∼3-log 10 compared to control animals. In nasal swabs, sgRNA declined 1-log 10 and virus remained culturable. Anamnestic antibody responses (590-fold increase) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. We immunized rhesus macaques at weeks 0 and 4 and assessed immune responses over one year in blood, upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID 50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody binding titers also decreased in bronchoalveolar lavage (BAL). Four days after challenge, virus was unculturable in BAL and subgenomic RNA declined ∼3-log 10 compared to control animals. In nasal swabs, sgRNA declined 1-log 10 and virus remained culturable. Anamnestic antibody responses (590-fold increase) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.