Biology subjects

Trehan, A.

Publications and source records attributed to Trehan, A..

7 recordsLinked to original sources

bioRxiv2026

Comparative Single-Cell Profiling of CRISPR Knockout and Interference Defines Modality-Specific Strengths in Functional Genomics

Pooled CRISPR screens coupled with single-cell RNA sequencing enable high-throughput functional interrogation of gene regulatory networks, yet systematic comparisons of CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) remain limited. We established a single-cell CRISPRko workflow and benchmarked it against CRISPRi using 87 sgRNAs targeting 29 unfolded protein response genes. CRISPRko generated transcriptional phenotypes are highly concordant with CRISPRi, and induced comparable pathway-level responses. While CRISPRi allows direct assessment of target gene repression, CRISPRko provides an effective complementary approach for complete loss-of-function studies, expanding the toolkit for single-cell functional genomics.

genomics↗

bioRxiv2026

Changes in perineuronal net and parvalbumin expression in the orbitofrontal cortex of male Wistar rats following repeated fentanyl administration

The misuse of opioid medications is a significant health issue in the United States. Very few studies have investigated the effect of opioids on perineuronal nets (PNNs), scaffold-like structures that surround neurons and are involved in the regulation of plasticity-dependent mechanisms such as development, learning and memory, and acquisition of addiction-like phenotypes. Regulation of PNNs in the orbitofrontal cortex (OFC) during periods of drug intoxication or withdrawal is widely unknown. In this study, male Wistar rats were injected with fentanyl (0.125 mg/kg, s.c.) or 0.9% saline twice daily for 7 days and once on day 8 (7continuous days following by 3 days of abstinence) or twice daily for 15 days (5 continuous days followed by 2 days of abstinence for more than 3 weeks) and twice on day 16. Antinociception was evaluated using the tail immersion test immediately before and 30 minutes after injections. Whole-brain coronal slices were collected, and immunohistochemistry was used to identify Wisteria Floribunda Agglutinin (WFA)-positive PNNs and parvalbumin (PV)-expressing cells. Results confirmed that repeated fentanyl injections induced tolerance to the antinociceptive effects, which normalized following acute abstinence periods. WFA intensity decreased following 8 days of injections. Analyses confirmed significant correlations between PV+ density and tail withdrawal latency following 8 days of fentanyl injections. These data confirm that repeated fentanyl injections modulate both WFA+ and PV+ expression in the rodent brain and antinociceptive tolerance in a duration-dependent manner. Overall, these data suggest that perineuronal nets may mediate opioid-induced behavioral effects, such as antinociceptive tolerance, following repeated administration and abstinence in rats.

neuroscience↗

bioRxiv2026

Decoding the MYC locus reveals a druggable ultraconserved RNA element

The human genome is dominated by noncoding sequences, most of which are poorly conserved across species. How genetic information is distributed between coding and noncoding regions remains a fundamental unresolved question. Using CRISPR saturation mutagenesis at base-pair resolution, we mapped the functional fitness landscape of the 10-kb human MYC locus with a near-PAMless, high-fidelity SpRY-Cas9. This unbiased interrogation revealed that the majority (67%) of functionally essential base-pairs in this locus are noncoding. Paradoxically, the phenotypic impact of noncoding sequences correlates inversely with evolutionary conservation, driven in part by rapidly diverging cis-regulatory DNA elements that remain functionally constrained in humans. Within this landscape, we identified an ultraconserved RNA element in the 3 untranslated region (UTR) that is indispensable for MYC-dependent cancer cells. Remarkably, steric-blocking antisense oligos targeting this RNA element selectively eliminate MYC-addicted cancer cells by suppressing MYC function without reducing MYC abundance. Mechanistically, this 3' UTR element promotes perinuclear localization of MYC mRNA and efficient nuclear import of the short-lived MYC protein, enabling its function as a nuclear transcription factor. Together, these findings highlight noncoding sequences as major carriers of functional genetic information, provide a comprehensive fitness map of the MYC locus, and uncover a therapeutically actionable RNA element that disables MYC-driven cancer.

genomics↗

bioRxiv2021

RNA-guided cell targeting with CRISPR/RfxCas13d collateral activity in human cells

While single-cell sequencing has allowed rapid identification of novel cell types or states and associated RNA markers, functional studies remain challenging due to the lack of tools that are able to target specific cells based on these markers. Here we show that targeting a single marker RNA with CRISPR/RfxCas13d led to collateral transcriptome destruction in human cells, which can be harnessed to inhibit cell proliferation or to suppress cell state transition.

molecular biology↗

bioRxiv2021

The 22q11.2 region regulates presynaptic gene-products linked to schizophrenia

To study how the 22q11.2 deletion predisposes to psychiatric disease, we generated induced pluripotent stem cells from deletion carriers and controls, as well as utilized CRISPR/Cas9 to introduce the heterozygous deletion into a control cell line. Upon differentiation into neural progenitor cells, we found the deletion acted in trans to alter the abundance of transcripts associated with risk for neurodevelopmental disorders including Autism Spectrum Disorder. In more differentiated excitatory neurons, altered transcripts encoded presynaptic factors and were associated with genetic risk for schizophrenia, including common (per-SNP heritability p ({tau}c)= 4.2 x 10-6) and rare, loss of function variants (p = 1.29x10-12). These findings suggest a potential relationship between cellular states, developmental windows and susceptibility to psychiatric conditions with different ages of onset. To understand how the deletion contributed to these observed changes in gene expression, we developed and applied PPItools, which identifies the minimal protein-protein interaction network that best explains an observed set of gene expression alterations. We found that many of the genes in the 22q11.2 interval interact in presynaptic, proteasome, and JUN/FOS transcriptional pathways that underlie the broader alterations in psychiatric risk gene expression we identified. Our findings suggest that the 22q11.2 deletion impacts genes and pathways that may converge with risk loci implicated by psychiatric genetic studies to influence disease manifestation in each deletion carrier.

neuroscience↗

bioRxiv2021

Astrocytic cell adhesion genes linked to schizophrenia promote synaptic programs in neurons

The maturation of neurons and the development of synapses - while emblematic of neurons - also rely on interactions with astrocytes and other glia. To study the role of glia-neuron interactions, we analyzed the transcriptomes of human pluripotent stem cell (hPSC)-derived neurons, from a total of 80 human donors, that were cultured with or without contact with glial cells. We found that the presence of astrocytes enhanced synaptic gene-expression programs in neurons. These changes in neuronal synaptic gene expression correlated with increased expression in the co-cultured glia of genes that encode synaptic cell adhesion molecules, and they were greatly enhanced in the glia in coculture. Both the neuronal and astrocyte gene-expression programs were enriched for genes that are linked to schizophrenia risk. Physical contact between the two cell types was required for the induction of synaptic programs in neurons. Our results suggest that astrocyte-expressed genes with synaptic functions are associated with stronger expression of synaptic genetic programs in neurons and suggest a potential role for astrocyte-neuron interactions in schizophrenia.

molecular biology↗

bioRxiv2019

Massive parallel variant characterization identifies NUDT15 alleles associated with thiopurine toxicity

As a prototype of genomics-guided precision medicine, individualized thiopurine dosing based on pharmacogenetics is a highly effective way to mitigate hematopoietic toxicity of this class of drugs. Recently, NUDT15 deficiency was identified as a novel genetic cause of thiopurine toxicity, and NUDT15-informed preemptive dose reduction is quickly adopted in clinical settings. To exhaustively identify pharmacogenetic variants in this gene, we developed massively parallel NUDT15 function assays to determine variants effect on protein abundance and thiopurine cytotoxicity. Of the 3,097 possible missense variants, we characterized the abundance of 2,922 variants and found 54 hotspot residues at which variants resulted in complete loss of protein stability. Analyzing 2,935 variants in the thiopurine cytotoxicity-based assay, we identified 17 additional residues where variants altered NUDT15 activity without affecting protein stability. We identified structural elements key to NUDT15 stability and/or catalytical activity with single amino-acid resolution. Functional effects for NUDT15 variants accurately predicted toxicity risk alleles in 2,398 patients treated with thiopurines, with 100% sensitivity and specificity, in contrast with poor performance of bioinformatic prediction algorithms. In conclusion, our massively parallel variant function assays identified 1,103 deleterious NUDT15 variants, providing a comprehensive reference of variant function and vastly improving the ability to implement pharmacogenetics-guided thiopurine treatment individualization.

genomics↗