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gene therapy for treatment of Inborn errors of Immunity (IEIs) have demonstrated significant clinical benefit in multiple Phase I/II clinical trials. Current approaches rely on engineered retroviral vectors to randomly integrate copy(s) of gene-of-interest in autologous hematopoietic stem/progenitor cells (HSPCs) genome permanently to provide gene function in transduced HSPCs and their progenies. To circumvent concerns related to potential genotoxicities due to the random vector integrations in HSPCs, targeted correction with CRISPR-Cas9-based genome editing offers improved precision for functional correction of multiple IEIs. We compare the two approaches for integration of transgene for functional correction of HSPCs from patients with X-linked Severe Combined Immunodeficiency (SCID-X1 or XSCID); delivery current clinical lentivector (LV)- versus targeted insertion (TI) of homology-directed repair (HDR) when using an adeno-associated virus (AAV)- donor following double-strand DNA break at the endogenous locus. differentiation of LV- or TI-treated XSCID HSPCs similarly overcome differentiation block into Pre-T-I and Pre-T-II lymphocytes but we observed significantly superior development of NK cells when corrected by TI (40.7% versus 4.1%, p = 0.0099). Transplants into immunodeficient mice demonstrated robust engraftment (8.1% and 23.3% in bone marrow) for LV- and TI- HSPCs with efficient T cell development following TI- in all four patients' HSPCs. Extensive specificity analysis of CRISPR-Cas9 editing with rhAmpSeq covering 82 predicted off-target sites found no evidence of indels in edited cells before ( ) or following transplant, in stark contrast to LV's non-targeted vector integration sites. Together, the improved efficiency and safety of correction CRISPR-Cas9-based TI approach provides a strong rationale for a clinical trial for treatment of XSCID patients.

Engineering and characterizing proteins can be time-consuming and cumbersome, motivating the development of generalist CRISPR–Cas enzymes 1 , 2 , 3 – 4 to enable diverse genome-editing applications. However, such enzymes have caveats such as an increased risk of off-target editing 3 , 5 , 6 . Here, to enable scalable reprogramming of Cas9 enzymes, we combined high-throughput protein engineering with machine learning to derive bespoke editors that are more uniquely suited to specific targets. Through structure–function-informed saturation mutagenesis and bacterial selections, we obtained nearly 1,000 engineered SpCas9 enzymes and characterized their protospacer-adjacent motif (PAM) 7 requirements to train a neural network that relates amino acid sequence to PAM specificity. By utilizing the resulting PAM machine learning algorithm (PAMmla) to predict the PAMs of 64 million SpCas9 enzymes, we identified efficacious and specific enzymes that outperform evolution-based and engineered SpCas9 enzymes as nucleases and base editors in human cells while reducing off-targets. An in silico-directed evolution method enables user-directed Cas9 enzyme design, including for allele-selective targeting of the RHO P23H allele in human cells and mice. Together, PAMmla integrates machine learning and protein engineering to curate a catalogue of SpCas9 enzymes with distinct PAM requirements, motivating a shift away from generalist enzymes towards safe and efficient bespoke Cas9 variants. Combined high-throughput protein engineering with machine learning to curate libraries of CRISPR genome-editing enzymes with distinct genome targeting properties is described.

X-linked chronic granulomatous disease is an immunodeficiency characterized by defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the 13 exons and splice sites of the CYBB gene, resulting in loss of gp91phox protein. Here we report gene correction by homology-directed repair in patient hematopoietic stem/progenitor cells (HSPCs) using CRISPR/Cas9 for targeted insertion of CYBB exon 1–13 or 2–13 cDNAs from adeno-associated virus donors at endogenous CYBB exon 1 or exon 2 sites. Targeted insertion of exon 1–13 cDNA did not restore physiologic gp91phox levels, consistent with a requirement for intron 1 in CYBB expression. However, insertion of exon 2–13 cDNA fully restored gp91phox and ROS production upon phagocyte differentiation. Addition of a woodchuck hepatitis virus post-transcriptional regulatory element did not further enhance gp91phox expression in exon 2–13 corrected cells, indicating that retention of intron 1 was sufficient for optimal CYBB expression. Targeted correction was increased ~1.5-fold using i53 mRNA to transiently inhibit nonhomologous end joining. Following engraftment in NSG mice, corrected HSPCs generated phagocytes with restored gp91phox and ROS production. Our findings demonstrate the utility of tailoring donor design and targeting strategies to retain regulatory elements needed for optimal expression of the target gene.

Preformed transposase-transposon complexes called 'Transposomes' have been electroporated into bacterial cells. The magnesium dependent process of insertion of the transposable element into bacterial chromosomal DNA occurs in vivo. The transposition efficiency of a Transposome containing a kanamycin marker was between 1.0 x 10(4) and 1.0 x 10(7) kanamycin resistant clones per microgram of transposon DNA in three gram-negative enteric bacterial species. Transposon integration sites were examined by direct genome sequencing of chromosomal DNA. Genomic DNA was isolated from transposition clones and directly cycle sequenced with primers specific for the ends of the transposon. The precise location of genome interruption for a transposition clone was identified by homology to known genes or sequences. Mutant phenotypes were rapidly correlated with genomic insertions sites.

Among a large cohort of women with viable singleton pregnancies who underwent elective repeat cesarean sections, more than a third of deliveries were performed before 39 weeks of gestation. As compared with deliveries at or after 39 weeks, deliveries before 39 weeks of gestation — even those during the last 3 days before week 39 — were associated with an increased risk of a composite primary outcome that included neonatal death, respiratory complications, need for mechanical ventilation, treated hypoglycemia, newborn sepsis, and admission to the neonatal intensive care unit. As compared with deliveries at or after 39 weeks, cesarean deliveries before 39 weeks of gestation were associated with an increased risk of a composite primary outcome that included neonatal death, respiratory complications, need for mechanical ventilation, treated hypoglycemia, newborn sepsis, and admission to the neonatal intensive care unit. Infants born before 39 weeks of gestation are at increased risk for neonatal adverse respiratory outcomes, and the risk increases progressively as gestational age at birth declines. 1 , 2 Thus, prelabor elective delivery (delivery in the absence of a specific maternal or fetal indication) is proscribed before 39 weeks unless fetal lung maturity has been demonstrated. 3 , 4 As compared with infants born vaginally, those born by cesarean section are at increased risk for adverse respiratory outcomes, especially when delivery occurs before the onset of labor. 1 , 2 , 5 – 11 This increased risk persists even in infants who are delivered by cesarean section . . .

Women with a history of preterm delivery are at high risk for recurrence in subsequent pregnancies, and an effective strategy to reduce this risk has been lacking. In this randomized, placebo-controlled trial, weekly injections of 17 alpha-hydroxyprogesterone caproate reduced the risk of delivery before 37 weeks of gestation by one third among such high-risk women. Injections of 17 alpha-hydroxyprogesterone reduced the risk among high-risk women. Preterm delivery — that is, delivery before 37 completed weeks of gestation — is the major determinant of infant mortality in developed countries. 1 Preterm delivery is more common in the United States than in many other developed countries and is the factor most responsible for the relatively high infant mortality in this country. 1 The rate of preterm delivery in the United States has increased progressively from 9 percent to 12 percent over the past two decades. 2 Despite many trials of reduced activity, tocolytic therapy, antibiotic therapy, and other strategies for prevention, no effective and reproducible method of preventing preterm delivery . . .

In this multicenter, four-year observational study of women with a history of cesarean section and a singleton gestation, a trial of labor was associated with a higher risk of symptomatic uterine rupture in the mother and hypoxic–ischemic encephalopathy in the infant than was elective cesarean delivery, although the absolute risks of these complications were low. The findings from this study should help inform women about their choices regarding the type of delivery after a prior cesarean section. The findings from this study should help inform women about their choices regarding the type of delivery after a prior cesarean section. The overall rate of cesarean delivery in the United States has risen dramatically, from 5 percent of all deliveries in 1970 to a high of 26 percent in 2002. 1 Efforts to reduce the number of cesarean births, although initially successful, failed to achieve the U.S. Public Health Service goals, set in 1990. These goals included achieving an overall rate of cesarean delivery of 15 percent, and a rate of vaginal birth after previous cesarean section of 35 percent of deliveries after previous cesarean sections, by the year 2000. 2 The Healthy People 2010 report published in 2000 proposes a target rate . . .

DNA transposition is an important biological phenomenon that mediates genome rearrangements, inheritance of antibiotic resistance determinants, and integration of retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with applications in creating insertional knockout mutations, generating gene–operon fusions to reporter functions, providing physical or genetic landmarks for the cloning of adjacent DNAs, and locating primer binding sites for DNA sequence analysis. DNA transposition studies to date usually have involved strictly in vivo approaches, in which the transposon of choice and the gene encoding the transposase responsible for catalyzing the transposition have to be introduced into the cell to be studied (microbial systems and applications are reviewed in ref. 1 ). However, all in vivo systems have a number of technical limitations. For instance, the transposase must be expressed in the target host, the transposon must be introduced into the host on a suicide vector, and the transposase usually is expressed in subsequent generations, resulting in potential genetic instability. A number of in vitro transposition systems (for Tn 5 , Tn 7 , Mu, Himar1 , and Ty 1 ) have been described, which bypass many limitations of in vivo systems 2 , 3 , 4 , 5 , 6 , 7 . For this purpose, we have developed a technique for transposition that involves the formation in vitro of released Tn 5 transposition complexes (Transposomes TM ) followed by introduction of the complexes into the target cell of choice by electroporation. In this report, we show that this simple, robust technology can generate high-efficiency transposition in all tested bacterial species ( Escherichia coli , Salmonella typhimurium , and Proteus vulgaris ) We also isolated transposition events in the yeast Saccharomyces cerevisiae .

Objective. We hypothesized that oligohydramnios would be diagnosed more frequently in the warm summer months when dehydration might be more common. Methods. The clinical diagnosis of oligohydramnios was extracted from the databases of four completed National Institute of Child Health and Human Development Maternal Fetal Medicine Units Network protocols. These data were stratified by quarter of delivery and compared using Fisher's Exact Test. Results. The clinical diagnosis of oligohydramnios was made more frequently in deliveries occurring in the summer months of June, July and August as compared with the remainder of the calendar year (7.2% vs 5.9%, p = 0.0178). Conclusions. In these studies the diagnosis of oligohydramnios is made more frequently in those pregnancies delivered during the summer months. Although not proven by this association, maternal dehydration may contribute to this finding.