A practical framework for isoform-level analysis in single-cell CRISPR screens is provided, identifying current capabilities and limitations, and providing a practical framework for isoform-level analysis in perturbation studies scale.
This review examines the experimental and computational foundations of scLR-seq, including platform selection, library design, cell barcode and unique molecular identifier recovery, transcript discovery, and isoform quantification, and summarize emerging insights into isoform usage, alternative splicing, transcription start and end site selection, allele-specific expression, fusion transcripts, transposable element-derived transcripts, and RNA modifications.
Retinal development has been extensively studied and key transcriptional regulators that drive fate decisions have been identified for major cell classes. These findings were confirmed and deepened in recent years with the advance of single cell RNA sequencing (Scrase). However, many processes that guide progenitor to postmitotic cell differentiation remain elusive, especially since some genes seem to yield different cell populations without apparent correlation with expression level or timing. Here, differential transcript isoform usage might play a role in diversifying the function of developmental genes. In short-read based scRNAseq, isoforms can only be identified if a read maps to a unique sequence or exon junction. However, due to the sparsity and very short reads, these events are extremely rare. We combined a commercial scRNAseq kit, that produces barcoded, full-length cDNA with Oxford Nanopore Technologies based long-read sequencing to generate the first single cell long-read sequencing dataset of fetal human retina. It can help elucidate the role of alternative splicing in retinal development and guide the design of transcript-specific gene therapies for retinal regeneration.
Abstract Both genomic mutations and RNA editing contribute to functional complexity and drive adaptive evolution. Single-cell profiling offers deep insight into the cis-regulatory mechanisms underlying these variations. Using 13 025 single-cell Smart-Seq libraries from whole-body Drosophila melanogaster, we unexpectedly found that 94.0% of adenosine-to-inosine RNA editing sites and 92.8% of heterozygous single nucleotide polymorphismss (SNPs) with sufficient “unique fragment support” exhibit binary expression (0 or 1) in a single cell. The genotypes of representative heterozygous SNPs were validated by Sanger sequencing. Meanwhile, binary RNA editing itself is logically questionable due to elusive mechanism, compromised condition specificity, and untenable heterozygote advantage. This fact that for most cases in Smart-Seq, only a single allele (out of the various haplotypes) is finally maintained per cell, raises the following concern. Regardless of the biological or technical explanations like monoallelic transcriptional burst, dropout, or amplification bias that might account for this binary expression pattern, our findings conservatively indicate that Smart-Seq may not be good at analyzing molecular diversity and that the results need to be interpreted with caution.
Y. Duan, Jiyao Liu, Shiwen Xu et al.· Nucleic Acids Research· 0 citations
CRISPR interference (CRISPRi) is a powerful technology for studying loss-of-function phenotypes, enabling transient and reversible control of gene expression without the introduction of double-stranded DNA breaks. The cost of conducting large-scale CRISPR screens necessitates the selection of effective and specific single-guide RNAs for the design of compact libraries. While several genome-wide CRISPRi-Cas9 libraries have been created, updates to transcript annotations, the generation of higher-resolution chromatin accessibility datasets, and the development of newer on-target prediction models motivate an updated CRISPRi library design approach. Here, we generate large CRISPRi datasets tiling essential and nonessential genes. We compare the performance of multiple KRAB domain systems, develop an updated CRISPRi-specific on-target scoring scheme, and quantitatively characterize off-target effects associated with seed-sequence patterns. We leverage these findings to design an optimized CRISPRi-Cas9 library, Katsano, and validate its performance with genome-wide viability screens.
Smriti Srikanth, Fengyi Zheng, Laura M Drepanos et al.· Cell Genomics· 0 citations
Single-cell RNA sequencing has transformed our understanding of cellular systems, yet the reliance on short-read sequencing restricts analysis to gene-level quantification and obscures the immense biological diversity generated by alternative splicing. While long-read sequencing technologies can capture full-length RNA and resolve transcript isoforms, current platforms remain constrained by throughput and high per-base costs, rendering them impractical for modern million-cell applications. To address this critical limitation, we developed and optimized sequencing-by-expansion (SBX) chemistry for high-throughput single-cell RNA isoform profiling. Integrated within the AXELIOS 1 sequencing platform, SBX employs a unique biochemical conversion process that transforms complementary DNA into expanded surrogate high signal-to-noise polymers called Xpandomers which are sequenced via translocation through a dense nanopore array yielding over 9.5 billion reads in a two-hour run. To leverage this unique data type for long-read single-cell RNA isoform sequencing, we developed the Consensus UMI Deduplication using Longest Length (CUDLL) algorithm, which computationally consolidates variable-length raw SBX reads into single, high-fidelity consensus reads, elevating sequence accuracy to 99.83% and maximizing per transcript read length. We demonstrate that this consensus approach successfully captures the vast isoform diversity of single-cell libraries and enables the accurate measure of differential isoform expression across distinct cell types in peripheral blood mononuclear cells. Furthermore, SBX coupled with CUDLL efficiently resolves T-cell and B-cell receptor clonotypes directly from whole-transcriptome libraries without the need for VDJ-specific target enrichment. Ultimately, this work establishes SBX and the AXELIOS 1 as a transformative platform for high-scale single-cell isoform sequencing.
Christophe H. Georgescu, Ghamdan Al-Eryani, Allison Brookhart et al.· bioRxiv· 0 citations
This work presents CRISPRLungo, a computational pipeline specifically designed for long-read amplicon sequencing of gene edited samples that incorporates unique molecular identifier-based error correction and statistical filtering to distinguish true editing events from background noise, enabling robust detection of small indels and structural variants.
Gue-Ho Hwang, Benjamin Vyshedskiy, Timothy M. Barry et al.· Nature Biomedical Engineerin...· 0 citations