A5067984297- CRISPR and Genetic Engineering
- CAR-T cell therapy research
- RNA and protein synthesis mechanisms
- Animal Genetics and Reproduction
- Genomics and Phylogenetic Studies
- RNA modifications and cancer
- Evolution and Genetic Dynamics
- Viral Infectious Diseases and Gene Expression in Insects
- Virus-based gene therapy research
- Cardiac electrophysiology and arrhythmias
- Advanced biosensing and bioanalysis techniques
- RNA Research and Splicing
- Renal Transplantation Outcomes and Treatments
- Immune Cell Function and Interaction
- Bacterial Genetics and Biotechnology
- Chromosomal and Genetic Variations
- Single-cell and spatial transcriptomics
- Antibiotics Pharmacokinetics and Efficacy
- Fungal Biology and Applications
- Cancer Genomics and Diagnostics
- Amyloidosis: Diagnosis, Treatment, Outcomes
- Organ Transplantation Techniques and Outcomes
- Immunotherapy and Immune Responses
- Nanowire Synthesis and Applications
- Antifungal resistance and susceptibility
University of California, San Francisco
2009-2025
Parker Institute for Cancer Immunotherapy
2021-2025
Gladstone Institutes
2021-2025
City College of San Francisco
2023-2025
Harvard University Press
2023
Universidad Católica de Santa Fe
2022
Boston Children's Hospital
2021
Innovative Genomics Institute
2019-2020
University of California, Berkeley
2019-2020
BioTelemetry (United States)
2016-2019
We describe the construction and characterization of a genomically recoded organism (GRO). replaced all known UAG stop codons in Escherichia coli MG1655 with synonymous UAA codons, which permitted deletion release factor 1 reassignment translation function. This GRO exhibited improved properties for incorporation nonstandard amino acids that expand chemical diversity proteins vivo. The also increased resistance to T7 bacteriophage, demonstrating new genetic codes could enable viral resistance.
Exploiting Redundancy The genetic code is redundant—multiple codons can for the same amino acid. So-called synonymous codon changes within genes nonetheless have substantial affects on protein expression, which been attributed to in structure of 5′ messenger RNAs, among other factors. Goodman et al. (p. 475 , published online 26 September) built and measured expression a synthetic library 14,000 variant N-terminal sequences 137 Escherichia coli show that, unexpectedly, rare had bigger effect...
We present genome engineering technologies that are capable of fundamentally reengineering genomes from the nucleotide to megabase scale. used multiplex automated (MAGE) site-specifically replace all 314 TAG stop codons with synonymous TAA in parallel across 32 Escherichia coli strains. This approach allowed us measure individual recombination frequencies, confirm viability for each modification, and identify associated phenotypes. developed hierarchical conjugative assembly (CAGE) merge...
The inability to predict heterologous gene expression levels precisely hinders our ability engineer biological systems. Using well-characterized regulatory elements offers a potential solution only if such behave predictably when combined. We synthesized 12,563 combinations of common promoters and ribosome binding sites simultaneously measured DNA, RNA, protein from the entire library. simple model, we found that RNA were within twofold expected 80% 64% time, respectively. large dataset...
Recoding and repurposing genetic codons By recoding bacterial genomes, it is possible to create organisms that can potentially synthesize products not commonly found in nature. systematic replacement of seven with synonymous alternatives for all protein-coding genes, Ostrov et al. recoded the Escherichia coli genome. The number E. code was reduced from 64 57 by removing instances UAG stop codon excising two arginine codons, leucine serine codons. Over 90% functionality successfully retained....
Zoonomia is the largest comparative genomics resource for mammals produced to date. By aligning genomes 240 species, we identify bases that, when mutated, are likely affect fitness and alter disease risk. At least 332 million (~10.7%) in human genome unusually conserved across species (evolutionarily constrained) relative neutrally evolving repeats, 4552 ultraconserved elements nearly perfectly conserved. Of 101 significantly constrained single bases, 80% outside protein-coding exons half...
Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing nature envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses scale specificity of CRISPR-Cas methods. We use targeted reverse-transcription activity retrons produce single-stranded DNA (ssDNA) vivo, incorporating edits at >90% efficiency enabling multiplexed...
Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells refractory cancers but have shown limited efficacy in persistent, recurrent malignancies. Here, we introduce “CAR Pooling,” a multiplexed approach rapidly identify CAR designs with clinical potential. Forty CARs domains derived from range of immune cell lineages were evaluated pooled assays for their ability stimulate critical effector functions during repetitive stimulation that mimics long-term...
Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers synthetic knockin (KI) sequences reprogram functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for construction DNA libraries using barcoded multicistronic adaptors. We built two ModPoKI 100 transcription factors (TFs) 129 natural surface receptors (SRs). Over 30 screens across human TCR- CAR-T cells...
Objective A clinical trial was conducted in healthy adult volunteers to assess the effect of levofloxacin, moxifloxacin, and ciprofloxacin on QT QTc interval. Methods Electrocardiograms were recorded 24 hours before after subjects took placebo, 1000 mg 800 1500 a double-blind, randomized, 4-period, 4-treatment,4-sequence crossover trial. Changes interval from baseline assessed by several different methods. Results Increases compared with placebo consistently greater moxifloxacin either...
Recent proliferation of low-cost DNA sequencing techniques will soon lead to an explosive growth in the number sequenced genomes and turn manual annotations into a luxury. Mass spectrometry recently emerged as valuable technique for proteogenomic that improves on state-of-the-art predicting genes other features. However, previous approaches were limited single genome did not take advantage analyzing mass data from multiple at once. We show such comparative proteogenomics approach (like...
Pathogenic microbes exist in dynamic niches and have evolved robust adaptive responses to promote survival their hosts. The major fungal pathogens of humans, Candida albicans glabrata, are exposed a range environmental stresses hosts including osmotic, oxidative nitrosative stresses. Significant efforts been devoted the characterization each these In wild, cells frequently simultaneously combinations yet effects such combinatorial not explored. We developed common experimental platform...
Precise editing is essential for biomedical research and gene therapy. Yet, homology-directed genome modification limited by the requirements genomic lesions, homology donors endogenous DNA repair machinery. Here we engineered programmable cytidine deaminases test if could introduce site-specific to thymidine transitions in absence of targeted lesions. Our effectively convert specific cytidines thymidines with 13% efficiency Escherichia coli 2.5% human cells. However, off-target deaminations...
DNAplotlib ( www.dnaplotlib.org ) is a computational toolkit for the programmable visualization of highly customizable, standards-compliant genetic designs. Functions are provided to aid with both tasks and extract overlay associated experimental data. High-quality output produced in form vector-based PDFs, rasterized images, animated movies. All aspects rendering process can be easily customized or extended by user cover new forms part regulation. supports improved communication design...
Significance This work presents the genome-wide replacement of all rare AGR (AGA and AGG) arginine codons in essential genes Escherichia coli with synonymous CGN alternatives. Synonymous codon substitutions can lethally impact noncoding function by disrupting mRNA secondary structure ribosomal binding site-like motifs. Here we quantitatively define range tolerable deviation these metrics use this relationship to provide critical insight into choice recoded genomes. demonstrates that removal...
Abstract Engineering T cell specificity and function at multiple loci can generate more effective cellular therapies, but current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here we develop a one-step process to enrich unlabeled cells containing knock-ins target using family repair templates named synthetic exon expression disruptors (SEEDs). SEEDs associate transgene integration with the disruption paired endogenous surface protein while preserving in...
Abstract Adoptive immune cell therapies, such as CAR T, have revolutionized how we can approach cancer therapy, however challenges remain to increase their efficacy, response rates, and resistance dysfunction. The systematic investigation of gene programs governing human T function would be advantageous for reprogramming products clinical benefit. Recent studies demonstrated the power CRISPR-based forward genetic screens in primary cells nominate candidate perturbations enhanced function,...
Abstract Selection has been invaluable for genetic manipulation, although counter-selection historically exhibited limited robustness and convenience. TolC, an outer membrane pore involved in transmembrane transport E. coli, implemented as a selectable/counter-selectable marker, but escape frequency using colicin E1 precludes tolC inefficient manipulations and/or with large libraries. Here, we leveraged unbiased deep sequencing of 96 independent lineages exhibiting to identify...
We present a method for identifying genomic modifications that optimize complex phenotype through multiplex genome engineering and predictive modeling. apply our to identify six single nucleotide mutations recover 59% of the fitness defect exhibited by 63-codon E. coli strain C321.∆A. By introducing targeted combinations changes in we generate rich genotypic phenotypic diversity characterize clones using whole-genome sequencing doubling time measurements. Regularized multivariate linear...