Biology subjects

Kalhor, R.

Publications and source records attributed to Kalhor, R..

12 recordsLinked to original sources

bioRxiv2026

Analysis of sperm processing and cryopreservation procedures in the common marmoset (Callithrix jacchus) to reduce fragmentation and maximize total and progressive motility

The marmoset has emerged as an ideal species for studying complex neurodevelopmental diseases, and IVF is required for gene-editing success. Reliable sperm processing is paramount to several IVF techniques, including traditional insemination. If not used immediately, thermal stress from cryopreservation diminishes sperm quality, requiring improved processing methods. Swim-up and gradient methods may limit adequate sperm quality, and the lack of a standardized freezing protocol often results in impaired post-thaw motility. Thus, reliable standards for sperm processing and cryopreservation in the marmoset are needed to support vital IVF processes. Collected semen underwent one of four pre-processing incubation steps before processing by swim-up, sucrose gradient, or a microfluidics assay. Sperm parameters were assessed using a Hamilton Thorne Computer Assisted Sperm Analysis (CASA) system. To evaluate cryopreservation, microfluidics-processed sperm were subjected to five protocols using different cryoprotectants and cooling methods. After thawing, sperm samples were analyzed with the CASA system and a DNA fragmentation assay. Incubation for 30 minutes at 38C in TYH media decreased sperm aggregation, and subsequent microfluidics resulted in a higher percentage of motile and progressively motile sperm than swim-up or gradient methods. Our optimized cryopreservation protocol consistently achieved at least 20% progressive motility, a key benchmark for usable marmoset sperm, while previously published or commercial methods were less consistent. The cryopreservation protocol outperformed other published methods, having the least significant change in post-thaw motility without causing DNA damage. These findings establish standardized methods for reliably procuring and storing sperm samples for IVF or husbandry in the common marmoset.

developmental biology↗

bioRxiv2026

Genomic repeats for single-cell molecular recording

Genomic recording enables transient biological signals to be indelibly captured through DNA alterations, creating a permanent record of cellular history retrievable by sequencing. However, current methods are limited by scarce writing space, typically targeting only one or a few amenable genomic sites and requiring large cell populations for signal reconstruction. Here, we establish Repeats for Genomic Recording (RGRs): sequences with up to 400 copies targetable by a single CRISPR guide RNA, readable with a common primer pair, and predicted to have minimal functional impact. We demonstrate that RGRs enable both signal deconvolution in single cells and high-resolution recording in cell populations. Individual RGR sites exhibit distinct response kinetics; thus, combining them improves recording resolution beyond what redundancy alone provides, analogous to diversity reception in wireless communication. We develop a computational pipeline for systematic RGR identification, revealing 15,000 to 25,000 candidates per species across human, mouse, and zebrafish, thereby markedly expanding recording capacity and enabling cell-type-specific applications. Finally, we validate RGRs in live mice by recording long-term immediate early gene activity across the brain following epilepsy induction. This work establishes genomic repeats as a high-capacity platform for single-cell molecular recording in vivo.

synthetic biology↗

bioRxiv2024

Deconvolving organogenesis in space and time via spatial transcriptomics in thick tissues

Organ development is guided by a space-time landscape that constraints cell behavior. This landscape is challenging to characterize for the hair follicle - the most abundant mini organ - due to its complex microscopic structure and asynchronous development. We developed 3DEEP, a tissue clearing and spatial transcriptomic strategy for characterizing tissue blocks up to 400 {micro}m in thickness. We captured 371 hair follicles at different stages of organogenesis in 1 mm3 of skin of a 12-hour-old mouse with 6 million transcripts from 81 genes. From this single time point, we deconvoluted follicles by age based on whole-organ molecular pseudotimes to animate a stop-motion 3D atlas of follicle development along its trajectory. We defined molecular stages for hair follicle organogenesis and characterized the order of emergence for its structures, differential signaling dynamics at its top and bottom, morphogen shifts preceding and accompanying structural changes, and series of structural changes leading to the formation of its canal and opening. We further found that hair follicle stem cells and their niche are established and stratified early in organogenesis, before the formation of the hair bulb. Overall, this work demonstrates the power of increased depth of spatial transcriptomics to provide a four-dimensional analysis of organogenesis.

developmental biology↗

bioRxiv2024

Identifying a gene signature of metastatic potential by linking pre-metastatic state to ultimate metastatic fate

Identifying the key molecular pathways that enable metastasis by analyzing the eventual metastatic tumor is challenging because the state of the founder subclone likely changes following metastatic colonization. To address this challenge, we labeled primary mouse pancreatic ductal adenocarcinoma (PDAC) subclones with DNA barcodes to characterize their pre-metastatic state using ATAC-seq and RNA-seq and determine their relative in vivo metastatic potential prospectively. We identified a gene signature separating metastasis-high and metastasis-low subclones orthogonal to the normal-to-PDAC and classical-to-basal axes. The metastasis-high subclones feature activation of IL-1 pathway genes and high NF-{kappa}B and Zeb/Snail family activity and the metastasis-low subclones feature activation of neuroendocrine, motility, and Wnt pathway genes and high CDX2 and HOXA13 activity. In a functional screen, we validated novel mediators of PDAC metastasis in the IL-1 pathway, including the NF-{kappa}B targets Fos and Il23a, and beyond the IL-1 pathway including Myo1b and Tmem40. We scored human PDAC tumors for our signature of metastatic potential from mouse and found that metastases have higher scores than primary tumors. Moreover, primary tumors with higher scores are associated with worse prognosis. We also found that our metastatic potential signature is enriched in other human carcinomas, suggesting that it is conserved across epithelial malignancies. This work establishes a strategy for linking cancer cell state to future behavior, reveals novel functional regulators of PDAC metastasis, and establishes a method for scoring human carcinomas based on metastatic potential.

cancer biology↗

bioRxiv2024

Post-translation digital data encoding into the genomes of mammalian cell populations

High resolution cellular signal encoding is critical for better understanding of complex biological phenomena. DNA-based biosignal encoders alter genomic or plasmid DNA in a signal dependent manner. Current approaches involve the signal of interest affecting a DNA edit by interacting with a signal specific promoter which then results in expression of the effector molecule (DNA altering enzyme). Here, we present the proof of concept of a biosignal encoding system where the enzyme terminal deoxynucleotidyl transferase (TdT) acts as the effector molecule upon directly interacting with the signal of interest. A template independent DNA polymerase (DNAp), TdT incorporates nucleotides at the 3 OH ends of DNA substrate in a signal dependent manner. By employing CRISPR-Cas9 to create double stranded breaks in genomic DNA, we make 3OH ends available to act as substrate for TdT. We show that this system can successfully resolve and encode different concentrations of various biosignals into the genomic DNA of HEK-293T cells. Finally, we develop a simple encoding scheme associated with the tested biosignals and encode the message "HELLO WORLD" into the genomic DNA of HEK-293T cells at a population level with 91% accuracy. This work demonstrates a simple and engineerable system that can reliably store local biosignal information into the genomes of mammalian cell populations.

synthetic biology↗

bioRxiv2023

Temporal recording of mammalian development and precancer

Key to understanding many biological phenomena is knowing the temporal ordering of cellular events, which often require continuous direct observations [1, 2]. An alternative solution involves the utilization of irreversible genetic changes, such as naturally occurring mutations, to create indelible markers that enables retrospective temporal ordering [3-8]. Using NSC-seq, a newly designed and validated multi-purpose single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo, while incorporating assigned cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during murine embryonic development and identified new intestinal epithelial progenitor states by their unique genetic histories. NSC-seq analysis of murine adenomas and single-cell multi-omic profiling of human precancers as part of the Human Tumor Atlas Network (HTAN), including 116 scRNA-seq datasets and clonal analysis of 418 human polyps, demonstrated the occurrence of polyancestral initiation in 15-30% of colonic precancers, revealing their origins from multiple normal founders. Thus, our multimodal framework augments existing single-cell analyses and lays the foundation for in vivo multimodal recording, enabling the tracking of lineage and temporal events during development and tumorigenesis.

developmental biology↗

bioRxiv2022

Quantitative fate mapping: Reconstructing progenitor field dynamics via retrospective lineage barcoding

Natural and induced somatic mutations that accumulate in the genome during development record the phylogenetic relationships of cells; however, whether these lineage barcodes can capture the dynamics of complex progenitor fields remains unclear. Here, we introduce quantitative fate mapping, an approach to simultaneously map the fate and quantify the commitment time, commitment bias, and population size of multiple progenitor groups during development based on a time-scaled phylogeny of their descendants. To reconstruct time-scaled phylogenies from lineage barcodes, we introduce Phylotime, a scalable maximum likelihood clustering approach based on a generalizable barcoding mutagenesis model. We validate these approaches using realistically-simulated barcoding results as well as experimental results from a barcoding stem cell line. We further establish criteria for the minimum number of cells that must be analyzed for robust quantitative fate mapping. Overall, this work demonstrates how lineage barcodes, natural or synthetic, can be used to obtain quantitative fate maps, thus enabling analysis of progenitor dynamics long after embryonic development in any organism.

developmental biology↗

bioRxiv2022

Massively parallel genomic perturbations with multi-target CRISPR reveal new insights on Cas9 activity and DNA damage responses at endogenous sites

We present an approach that combines a Cas9 that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing technologies to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of novel multi-target gRNAs (mgRNAs), degenerate gRNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, discovering rapid post-cleavage Cas9 departure and repair factor loading at PAM-proximal genomic DNA. Moreover, by bypassing confounding effects from gRNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and Cas9 cleavage is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double strand breaks with high temporal resolution, discovering the presence, extent (under 2 kb), and kinetics (~ 0.5 hr) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci.

cell biology↗

bioRxiv2019

Recording temporal data onto DNA with minutes resolution

Recording biological signals can be difficult in three-dimensional matrices, such as tissue. We present a DNA polymerase-based strategy that records temporal biosignals locally onto DNA to be read out later, which could obviate the need to extract information from tissue on the fly. We use a template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT) that probabilistically adds dNTPs to single-stranded DNA (ssDNA) substrates without a template. We show that in vitro, the dNTP-incorporation preference of TdT changes with the presence of Co2+, Ca2+, Zn2+ and temperature. Extracting the signal profile over time is possible by examining the dNTP incorporation preference along the length of synthesized ssDNA strands like a molecular ticker tape. We call this TdT-based untemplated recording of temporal local environmental signals (TURTLES). We show that we can determine the time of Co2+ addition to within two minutes over a 60-minute period. Further, TURTLES has the capability to record multiple fluctuations. We can estimate the rise and fall of an input Co2+ pulse to within three minutes. TURTLES has at least 200-fold better temporal resolution than all previous DNA-based recording techniques.

synthetic biology↗

bioRxiv2018

Enzymatic DNA synthesis for digital information storage

DNA is an emerging storage medium for digital data but its adoption is hampered by limitations of phosphoramidite chemistry, which was developed for single-base accuracy required for biological functionality. Here, we establish a de novo enzymatic DNA synthesis strategy designed from the bottom-up for information storage. We harness a template-independent DNA polymerase for controlled synthesis of sequences with user-defined information content. We demonstrate retrieval of 144-bits, including addressing, from perfectly synthesized DNA strands using batch-processed Illumina and real-time Oxford Nanopore sequencing. We then develop a codec for data retrieval from populations of diverse but imperfectly synthesized DNA strands, each with a ~30% error tolerance. With this codec, we experimentally validate a kilobyte-scale design which stores 1 bit per nucleotide. Simulations of the codec support reliable and robust storage of information for large-scale systems. This work paves the way for alternative synthesis and sequencing strategies to advance information storage in DNA.

synthetic biology↗

bioRxiv2018

A homing CRISPR mouse resource for barcoding and lineage tracing

Cellular barcoding using nuclease-induced genetic mutations is an effective approach that is emerging for recording biological information, including developmental lineages. We have previously introduced the homing CRISPR system as a promising methodology for generating such barcodes with scalable diversity and without crosstalk. Here, we present a mouse line (MARC1) with multiple genomically-integrated and heritable homing guide RNAs (hgRNAs). We determine the genomic locations of these hgRNAs, their activity profiles during gestation, and the diversity of their mutants. We apply the line for unique barcoding of mouse embryos and differential barcoding of embryonic tissues. We conclude that this mouse line can address the unique challenges associated with in vivo barcoding in mammalian model organisms and is thus an enabling platform for recording and lineage tracing applications in a mammalian model system.

synthetic biology↗

bioRxiv2017

Specific Virus-Host Genome Interactions Revealed By Tethered Chromosome Conformation Capture

Viruses have evolved a variety of mechanisms to interact with host cells for their adaptive benefits, including subverting host immune responses and hijacking host DNA replication/transcription machineries [1-3]. Although interactions between viral and host proteins have been studied extensively, little is known about how the vial genome may interact with the host genome and how such interactions could affect the activities of both the virus and the host cell. Since the three-dimensional organization of a genome can have significant impact on genomic activities such as transcription and replication, we hypothesize that such structure-based regulation of genomic functions also applies to viral genomes depending on their association with host genomic regions and their spatial locations inside the nucleus. Here, we used Tethered Chromosome Conformation Capture (TCC) to investigate viral-host genome interactions between the adenovirus and human lung fibroblast cells. We found viral-host genome interactions were enriched in certain active chromatin regions and chromatin domains marked by H3K27me3. The contacts by viral DNA seems to impact the structure and function of the host genome, leading to remodeling of the fibroblast epigenome. Our study represents the first comprehensive analysis of viral-host interactions at the genome structure level, revealing unexpectedly specific virus-host genome interactions. The non-random nature of such interactions indicates a deliberate but poorly understood mechanism for targeting of host DNA by foreign genomes.

microbiology↗