Biology subjects

Montalbano, A.

Publications and source records attributed to Montalbano, A..

4 recordsLinked to original sources

bioRxiv2025

Cutting Through the Artifacts: Dissecting gRNA Impurities with FUSS-seq

CRISPR-based therapeutics rely on guide RNAs (gRNAs) and the Cas9 endonuclease for precise gene editing. Ensuring gRNA purity and base-level sequence integrity is essential for clinical translation. While industry-standard practice relies on liquid chromatography-high-resolution mass spectrometry to assess oligonucleotide identity and purity, more recent FDA guidance recommends complementary base-by-base sequence analysis (FDA CBER Webinar, 2024). In this study, we evaluated next-generation sequencing (NGS) strategies for characterizing chemically synthesized gRNAs. We found that the widely used SMARTer assay, while capable of producing sequenceable libraries, introduced substantial artifacts during library preparation. These included truncated scaffold species at oligo(A) stretches in the scaffold region and 5'(n-1) deletions within the spacer sequence. Although absent in the original gRNA, these artifacts accounted for over 10% of the sequencing reads, creating the false appearance of impurities. Through experimental and computational approaches, we traced these artifacts to mispriming by template-switching oligonucleotides (TSOs). Importantly, these artifacts occur during sequencing, and although they do not reflect real gRNA impurities, they compromise assay accuracy and can obscure true sequence impurities. To overcome these limitations, we developed FUSS-seq (Full-length Uncoupled Second-strand Synthesis followed by sequencing), a novel assay that integrates principles from 5' RACE with a modified TSO bearing a 3' polymerase-blocking moiety. FUSS-seq markedly reduced artifacts and increased full-length gRNA recovery, providing a more accurate and lower-bias method for gRNA purity assessment. This approach supports improved Chemistry Manufacturing and Controls (CMC) characterization of gRNAs and strengthens the analytical toolkit needed for reliable CRISPR-based therapeutic development.

molecular biology↗

bioRxiv2023

LKB1 suppresses growth and promotes the internalization of EGFR through the PIKFYVE lipid kinase

The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1- mediated growth suppression we developed a spheroid-based cell culture assay to study LKB1- dependent growth. Using this assay, along with genome-wide CRISPR screens and validation with orthogonal methods, we discovered that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase, which promotes the internalization of wild-type EGFR. Our findings reveal a new mechanism of regulation of EGFR, which may have implications for the treatment of LKB1-mutant LUAD.

cancer biology↗

bioRxiv2020

Scalable pooled CRISPR screens with single-cell chromatin accessibility profiling

Pooled CRISPR screens have been used to identify genes responsible for specific phenotypes and diseases, and, more recently, to connect genetic perturbations with multi-dimensional gene expression profiles. Here, we describe a method to link genome-wide chromatin accessibility to genetic perturbations in single cells. This scalable, cost-effective method combines pooled CRISPR perturbations with a single-cell combinatorial indexing assay for transposase-accessible chromatin (CRISPR-sciATAC). Using a human and mouse species-mixing experiment, we show that CRISPR-sciATAC separates single cells with a low doublet rate. Then, in human myelogenous leukemia cells, we apply CRISPR-sciATAC to target 21 chromatin-related genes that are frequently mutated in cancer and 84 subunits and cofactors of chromatin remodeling complexes, generating chromatin accessibility data for ~30,000 single cells. Using this large-scale atlas, we correlate loss of specific chromatin remodelers with changes in accessibility -- globally and at the binding sites of individual transcription factors. For example, we show that loss of the H3K27 methyltransferase EZH2 leads to increased accessibility at heterochromatic regions involved in embryonic development and triggers expression of multiple genes in the HOXA and HOXD clusters. At a subset of regulatory sites, we also analyze dynamic changes in nucleosome spacing upon loss of chromatin remodelers. CRISPR-sciATAC is a high-throughput, low-cost single-cell method that can be applied broadly to study the role of genetic perturbations on chromatin in normal and disease states.

genomics↗

bioRxiv2018

Expanding the CITE-seq tool-kit: Detection of proteins, transcriptomes, clonotypes and CRISPR perturbations with multiplexing, in a single assay

Rapid technological progress in the recent years has allowed the high-throughput interrogation of different types of biomolecules from single cells. Combining several of these readouts into integrated multi-omic assays is essential to comprehensively understand and model cellular processes. Here, we report the development of Expanded CRISPR-compatible Cellular Indexing of Transcriptomes and Epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell: transcriptome, immune receptor clonotypes, surface markers, sample identity and sgRNAs. We demonstrate the use of ECCITE-seq to directly and efficiently capture sgRNA molecules and measure their effects on gene expression and protein levels, opening the possibility of performing high throughput single cell CRISPR screens with multimodal readout using existing libraries and commonly used vectors. Finally, by utilizing the combined phenotyping of clonotype and cell surface markers in immune cells, we apply ECCITE to study a lymphoma sample to discriminate cells and define molecular signatures of malignant cells within a heterogeneous population.

genomics↗