Refining chimeric antigen receptors via barcoded protein domain combination pooled screening.

Chimeric antigen receptor (CAR)-T cells represent a promising frontier in cancer immunotherapy. However, the current process for developing new CAR constructs is time consuming and inefficient. To address this challenge and expedite the evaluation and comparison of full-length CAR designs, we have devised a novel cloning strategy. This strategy involves the sequential assembly of individual CAR domains using blunt ligation, with each domain being assigned a unique DNA barcode. Applying this method, we successfully generated 360 CAR constructs that specifically target clinically validated tumor antigens CD19 and GD2. By quantifying changes in barcode frequencies through next-generation sequencing, we characterize CARs that best mediate proliferation and expansion of transduced T cells. The screening revealed a crucial role for the hinge domain in CAR functionality, with CD8a and IgG4 hinges having opposite effects in the surface expression, cytokine production, and antitumor activity in CD19- versus GD2-based CARs. Importantly, we discovered two novel CD19-CAR architectures containing the IgG4 hinge domain that mediate superior in vivo antitumor activity compared with the construct used in Kymriah, a U.S. Food and Drug Administration (FDA)-approved therapy. This novel screening approach represents a major advance in CAR engineering, enabling accelerated development of cell-based cancer immunotherapies.

Management of nutritional iron deficiency anemia for young children in the emergency department.

BACKGROUND: Guidelines for young children with nutritional iron deficiency anemia (IDA) presenting to the emergency department (ED) are lacking, leading to variability in care. We aimed to standardize management of these patients through the development and implementation of an evidence-based algorithm using quality improvement methodology. PROCEDURE: Baseline data of the target population (n = 42; 60% male; median age 22.5 months, median hemoglobin 5.3 g/dl) identified variability across four key measures of clinical management: laboratory evaluation, therapy choice, therapy administration, and patient disposition. Literature review and consensus from pediatric hematology providers informed a draft algorithm that was refined in an iterative multidisciplinary process. From September 2020 to June 2021, we aimed to increase IDA management per the algorithm by ≥20% relative to baseline for the four key outcome measures using sequential Plan-Do-Study-Act (PDSA) cycles. Process measures focusing on provider communication/documentation and balancing measures involving efficiency and therapy-related adverse events were assessed concurrently. RESULTS: Thirty-five patients were evaluated among four PDSA cycles and shared similar characteristics as the baseline population. Improvements of ≥20% above baseline adherence levels or 100% adherence were achieved for all outcome measure across four PDSA cycles. Adherence to recommended laboratory evaluation improved from 43 (baseline) to 71%, therapy choice from 78 to 100%, therapy administration from 50 to 83%, and disposition from 85 to 100%. ED length of stay remained stable. CONCLUSIONS: Implementation of a standardized algorithm for young children with nutritional IDA in the ED increased adherence to evidence-based patient care.

Reducing adverse impacts of Amazon hydropower expansion.

Proposed hydropower dams at more than 350 sites throughout the Amazon require strategic evaluation of trade-offs between the numerous ecosystem services provided by Earth's largest and most biodiverse river basin. These services are spatially variable, hence collective impacts of newly built dams depend strongly on their configuration. We use multiobjective optimization to identify portfolios of sites that simultaneously minimize impacts on river flow, river connectivity, sediment transport, fish diversity, and greenhouse gas emissions while achieving energy production goals. We find that uncoordinated, dam-by-dam hydropower expansion has resulted in forgone ecosystem service benefits. Minimizing further damage from hydropower development requires considering diverse environmental impacts across the entire basin, as well as cooperation among Amazonian nations. Our findings offer a transferable model for the evaluation of hydropower expansion in transboundary basins.

Repeatability and reproducibility of the pressure biofeedback unit.

INTRODUCTION: Although the pressure biofeedback unit (PBU) is used for muscular assessment and training, there is little evidence of its reproducibility and repeatability. OBJECTIVE: This study aims to assess intra- and inter-rater reproducibility and repeatability of the PBU in the assessment of the transverse abdominal (TrA), internal oblique (IO), low back multifidi, and deep neck flexors (DNF). METHODS: Fifty individuals had three muscular groups tested: TrA/IO, lower back multifidi, and DNF. For repeatability, one rater did three consecutive measures; for intra-rater reproducibility the same rater did two measures with seven-day intervals, and for inter-rater reproducibility, three raters, on the same day, did the measures. Data were analyzed with: Intraclass Correlation Coefficient (ICC), Standard Error of Measurement (SEM), and Minimal Detectable Change (MDC). (α = 0,05). RESULTS: Repeatability: TrA/IO (ICC = 0.847), Multifidi (ICC = 0.860), DNF (ICC = 0.831). Inter-rater reproducibility: TrA/IO (ICC = 0.876), Multifidi (ICC = 0.508), DNF (ICC = 0.442). Intra-rater reproducibility: TrA/IO (ICC = 0.747), Multifidi (ICC = 0.293), DNF (ICC = 0.685). Except for Multifidi, all the SEM values were less than 10 mmHg and the MDC values were less than 15 mmHg. CONCLUSIONS: The PBU can be used with reliability by different evaluators, although the evaluation of multifidi is not indicated.

Characterizing the portability of phage-encoded homologous recombination proteins.

Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded single-stranded DNA annealing proteins (SSAPs) improve HR 1,000-fold above endogenous levels. However, they are not broadly functional. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus and Caulobacter crescentus, we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host's single-stranded DNA-binding protein (SSB) and are portable between species only if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with SSBs can significantly improve genome editing efficiency, in some species enabling SSAP functionality even without host compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes that are inaccessible through sequential nucleotide conversions.

Improved bacterial recombineering by parallelized protein discovery.

Exploiting bacteriophage-derived homologous recombination processes has enabled precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, multiplex automated genome engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however, currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, efficient SSAPs must first be found. Here we introduce a high-throughput method for SSAP discovery that we call "serial enrichment for efficient recombineering" (SEER). By performing SEER in Escherichia coli to screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5- to 10-fold in E. coli, while PapRecT enables efficient recombineering in Pseudomonas aeruginosa, a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria.

T-Cell Lymphopenia Detected by Newborn Screening in Two Siblings with an Xq13.1 Duplication.

Newborn screening for severe combined immunodeficiency has proven successful in identifying infants with T-cell deficiencies before they become severely ill. Additionally, the newborn screen can detect subtle early phenotypes that may become severe later in life. We present the case of siblings with features suggestive of T-cell lymphopenia identified as having low T-cell receptor excision circles counts by newborn screening. Expanded immune testing showed robust lymphocyte mitogen and antigen responses with normal vaccine responses and immunoglobulin levels for both boys over time. Genetic analysis revealed an Xq13.1 duplication in each child not found in the mother. The variant is downstream of the IL2RG gene with potential regulatory significance, suggesting a mechanism for the T-cell lymphopenia. The newborn screen provided these patients heightened surveillance and patient-specific management, including delayed live vaccines and Pneumocystis jiroveci pneumonia prophylaxis. Fortunately, the brothers have not suffered invasive or opportunistic infections and are well at ages 3 and 4 years. In this report, we illustrate the challenges of managing seemingly asymptomatic immunodeficient patients without a definitive genetic diagnosis and show how unbiased genetic analysis can expand understanding about primary immunodeficiency phenotypes.

Efficient, footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies.

Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA), donor DNA and piggyBac transposase resulted in efficient, targeted genome editing and concurrent scarless transgene excision. Using this approach, in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%.

A general strategy to construct small molecule biosensors in eukaryotes.

Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.

Highly multiplexed subcellular RNA sequencing in situ.

Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.

Optimization of scarless human stem cell genome editing.

Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7-8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.

Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast.

Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers.

DNA built from modular repeats presents a challenge for gene synthesis. We present a solid surface-based sequential ligation approach, which we refer to as iterative capped assembly (ICA), that adds DNA repeat monomers individually to a growing chain while using hairpin 'capping' oligonucleotides to block incompletely extended chains, greatly increasing the frequency of full-length final products. Applying ICA to a model problem, construction of custom transcription activator-like effector nucleases (TALENs) for genome engineering, we demonstrate efficient synthesis of TALE DNA-binding domains up to 21 monomers long and their ligation into a nuclease-carrying backbone vector all within 3 h. We used ICA to synthesize 20 TALENs of varying DNA target site length and tested their ability to stimulate gene editing by a donor oligonucleotide in human cells. All the TALENS show activity, with the ones >15 monomers long tending to work best. Since ICA builds full-length constructs from individual monomers rather than large exhaustive libraries of pre-fabricated oligomers, it will be trivial to incorporate future modified TALE monomers with improved or expanded function or to synthesize other types of repeat-modular DNA where the diversity of possible monomers makes exhaustive oligomer libraries impractical.

Stable gene targeting in human cells using single-strand oligonucleotides with modified bases.

Recent advances allow multiplexed genome engineering in E. coli, employing easily designed oligonucleotides to edit multiple loci simultaneously. A similar technology in human cells would greatly expedite functional genomics, both by enhancing our ability to test how individual variants such as single nucleotide polymorphisms (SNPs) are related to specific phenotypes, and potentially allowing simultaneous mutation of multiple loci. However, oligo-mediated targeting of human cells is currently limited by low targeting efficiencies and low survival of modified cells. Using a HeLa-based EGFP-rescue reporter system we show that use of modified base analogs can increase targeting efficiency, in part by avoiding the mismatch repair machinery. We investigate the effects of oligonucleotide toxicity and find a strong correlation between the number of phosphorothioate bonds and toxicity. Stably EGFP-corrected cells were generated at a frequency of ~0.05% with an optimized oligonucleotide design combining modified bases and reduced number of phosphorothioate bonds. We provide evidence from comparative RNA-seq analysis suggesting cellular immunity induced by the oligonucleotides might contribute to the low viability of oligo-corrected cells. Further optimization of this method should allow rapid and scalable genome engineering in human cells.

Non-syndromic vestibular disorder with otoconial agenesis in tilted/mergulhador mice caused by mutations in otopetrin 1.

Otoconia are biominerals within the utricle and saccule of the inner ear that are critical for the perception of gravity and linear acceleration. The classical mouse mutant tilted (tlt) and a new allele, mergulhador (mlh), are recessive mutations that affect balance by impairing otoconial morphogenesis without causing collateral deafness. The mechanisms governing otoconial biosynthesis are not known. Here we show that tlt and mlh are mutant alleles of a novel gene (Otopetrin 1, Otop1), encoding a multi-transmembrane domain protein that is expressed in the macula of the developing otocyst. Both mutants carry single point mutations leading to non-conservative amino acid substitutions that affect two putative transmembrane (TM) domains (tlt, Ala(151)-->Glu in TM3; mlh, Leu(408)-->Gln in TM8). Otop1 and Otop1-like paralogues, Otop2 and Otop3, define a new gene family with homology to the C. elegans and D. melanoganster DUF270 genes.