Biology subjects

Aach, J.

Publications and source records attributed to Aach, J..

7 recordsLinked to original sources

bioRxiv2022

Iterative transcription factor screening enables rapid generation of microglia-like cells from human iPSC

The ability to differentiate stem cells into human cell types is essential to define basic mechanisms and therapeutics, especially for cell types not routinely accessible by biopsies. But while engineered expression of transcription factors (TFs) identified through TF screens has been found to rapidly and efficiently produce some cell types, generation of other cell types that require complex combinations of TFs has been elusive. Here we develop an iterative, pooled single-cell TF screening method that improves the identification of effective TF combinations using the generation of human microglia-like cells as a testbed: Two iterations identified a combination of SPI1, CEBPA, FLI1, MEF2C, CEBPB, and IRF8 as sufficient to differentiate human iPSC into microglia-like cells in 4 days. Characterization of TF-induced microglia demonstrated molecular and functional similarity to primary microglia. We explore the use of single-cell atlas reference datasets to confirm identified TFs and how combining single-cell TF perturbation and gene expression data can enable the construction of causal gene regulatory networks. We describe what will be needed to fashion these methods into a generalized integrated pipeline, further ideas for enhancement, and possible applications.

bioengineering↗

bioRxiv2020

Characterizing the portability of RecT-mediated oligonucleotide recombination

Bacterial genome editing methods are used to engineer strains for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but traditional techniques using endogenous RecA-mediated pathways are inefficient and laborious. Phage encoded RecT proteins can improve HR over 1000-fold, but these proteins have limited portability between species. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus, and Caulobacter crescentus we investigated the hostlimited functionality of RecTs. We find that these proteins specifically recognize the 7 C-terminal amino acids of the bacterial single-stranded DNA-binding protein (SSB), and are portable between species only if compatibility with this host domain is maintained. Furthermore, in some species, we find that co-expressing otherwise incompatible RecTs with a paired bacterial SSB is sufficient to establish functionality. Finally, we demonstrate that high-efficiency HR surpasses the mutational capacity of more widely used error-prone methods for genome diversification, and can be used to identify exceptional phenotypes inaccessible through sequential nucleotide conversions.

synthetic biology↗

bioRxiv2019

Efficient chromosome-scale haplotype-resolved assembly of human genomes

Haplotype-resolved or phased sequence assembly provides a complete picture of genomes and complex genetic variations. However, current phased assembly algorithms either fail to generate chromosome-scale phasing or require pedigree information, which limits their application. We present a method that leverages long accurate reads and long-range conformation data for single individuals to generate chromosome-scale phased assembly within a day. Applied to three public human genomes, PGP1, HG002 and NA12878, our method produced haplotype-resolved assemblies with contig NG50 up to 25 Mb and phased [~]99.5% of heterozygous sites to 98-99% accuracy, outperforming other approaches in terms of both contiguity and phasing completeness. We demonstrate the importance of chromosome-scale phased assemblies to discover structural variants (SVs), including thousands of new transposon insertions, and of highly polymorphic and medically important regions such as HLA and KIR. Our improved method will enable high-quality precision medicine and facilitate new studies of individual haplotype variation and population diversity.

bioinformatics↗

bioRxiv2019

A haplotype-aware de novo assembly of related individuals using pedigree graph

MotivationReconstructing high-quality haplotype-resolved assemblies for related individuals of various species has important applications in understanding Mendelian diseases along with evolutionary and comparative genomics. Through major genomics sequencing efforts such as the Personal Genome Project, the Vertebrate Genome Project (VGP), the Earth Biogenome Project (EBP) and the Genome in a Bottle project (GIAB), a variety of sequencing datasets from mother-father-child trios of various diploid species are becoming available.\n\nCurrent trio assembly approaches are not designed to incorporate long-read sequencing data from parents in a trio, and therefore require relatively high coverages of costly long-read data to produce high-quality assemblies. Thus, building a trio-aware assembler capable of producing accurate and chromosomal-scale diploid genomes in a pedigree, while being cost-effective in terms of sequencing costs, is a pressing need of the genomics community.\n\nResultsWe present a novel pedigree-graph-based approach to diploid assembly using accurate Illumina data and long-read Pacific Biosciences (PacBio) data from all related individuals, thereby generalizing our previous work on single individuals. We demonstrate the effectiveness of our pedigree approach on a simulated trio of pseudo-diploid yeast genomes with different heterozygosity rates, and real data from Arabidopsis Thaliana. We show that we require as little as 30x coverage Illumina data and 15x PacBio data from each individual in a trio to generate chromosomal-scale phased assemblies. Additionally, we show that we can detect and phase variants from generated phased assemblies.\n\nAvailabilityhttps://github.com/shilpagarg/WHdenovo\n\nContactshilpa_garg@hms.harvard.edu, gchurch@genetics.med.harvard.edu

bioinformatics↗

bioRxiv2018

Barcoded oligonucleotides ligated on RNA amplified for multiplex and parallel in-situ analyses

We present Barcoded Oligonucleotides Ligated On RNA Amplified for Multiplexed and parallel In-Situ analysis (BOLORAMIS), a reverse-transcription (RT)-free method for spatially-resolved, targeted, in-situ RNA identification of single or multiple targets. For this proof of concept, we have profiled 154 distinct coding and small non-coding transcripts ranging in sizes 18 nucleotides in length and upwards, from over 200, 000 individual human induced pluripotent stem cells (iPSC) and demonstrated compatibility with multiplexed detection, enabled by fluorescent in-situ sequencing. We use BOLORAMIS data to identify differences in spatial localization and cell-to-cell expression heterogeneity. Our results demonstrate BOLORAMIS to be a generalizable toolset for targeted, in-situ detection of coding and small non-coding RNA for single or multiplexed applications.

cell biology↗

bioRxiv2017

Toward D-Peptide Biosynthesis: Elongation Factor P Enables Ribosomal Incorporation Of Consecutive D-Amino Acids

To maintain stereospecific biochemistry in cells, living organisms have evolved mechanisms to exclude D-amino acids (DAA) in their protein synthesis machinery, which also limits our exploration of the realm of mirror-image molecules. Here, we show that high affinity between EF-Tu and aminoacyl-tRNA promotes D-amino acid incorporation. More strikingly, Elongation Factor P efficiently resolves peptidyl transferase stalling between two consecutive D-amino acids, and hence enables the translation of D-peptides.

biochemistry↗

bioRxiv2016

Optimizing complex phenotypes through model-guided multiplex genome engineering

Optimization of complex phenotypes in engineered microbial strains has traditionally been accomplished by laboratory evolution. However, only a subset of the resulting mutations may affect the phenotype of interest and many others may have unintended effects. Multiplexed genome editing can complement evolutionary approaches by creating diverse combinations of targeted changes, but in both cases it remains challenging to identify which alleles influence the desired phenotype. We present a method for identifying a minimal set of genomic modifications that optimizes a complex phenotype by combining iterative cycles of multiplex genome engineering and predictive modeling. We applied our method to the 63-codon E. coli strain C321.{Delta}A, which has 676 mutations relative to its wild-type ancestor, and identified six single nucleotide mutations that together recover 59% of the fitness defect exhibited by the strain. The resulting optimized strain, C321.DA.opt, is an improved chassis for production of proteins containing non-standard amino acids. Our data reveal how multiple cycles of multiplex automated genome engineering (MAGE) and inexpensive sequencing can generate rich genotypic and phenotypic diversity that can be combined with linear regression techniques to quantify individual allelic effects. While laboratory evolution relies on enrichment as a proxy for allelic effect, our model-guided approach is less susceptible than enrichment to bias from population dynamics and recombination efficiency. We also show that the method can identify beneficial de novo mutations that arise adventitiously. Beyond improving the fitness of C321, {Delta}A, our work provides a proof-of-principle for high-throughput quantification of individual allelic effects which can be used with any method for generating targeted genotypic diversity.

synthetic biology↗