However, the variable responses of patients with MLL-r in this trial underscore the need for additional mechanistic insights into functional regions of DOT1L to improve therapeutic efficacy and trial designs for DOT1L-targeted therapy. A selective DOT1L inhibitor, EPZ5676 (Pinometostat) 21, has demonstrated proof-of-principle clinical benefits via induction of differentiation of MLL-r leukemic cells in a phase I clinical trial 22. Recent achievements in cancer epigenetics include discovery of a central role for the H3K79 methyltransferase DOT1L in maintaining MLL-r leukemia, an aggressive malignancy recognized in 5–10% of human acute leukemia cases 19, 20. Furthermore, we couple the sc-Tiling with three-dimensional structural modeling and discovered a previously unrecognized self-regulatory domain in DOT1L that modulates the chromatin interaction, enzymatic activation, and therapeutic sensitivity in MLL-r leukemia. In this study, we develop a single-cell CRISPR gene tiling pipeline “sc-Tiling” to provide high-resolution transcriptomic profiling of the coding regions of histone H3 lysine 79 (H3K79) methyltransferase DOT1L, an epigenetic therapeutic candidate selectively essential to mixed-lineage leukemia gene-rearranged ( MLL-r) leukemia 17, 18, 19. Nevertheless, the potential of single-cell CRISPR screen technology to examine the gene function at a sub-gene resolution has not been fully explored. The current single-cell CRISPR screens focused on observing single-gene function, gene-to-gene interaction, and enhancer-to-gene regulation 10, 12, 13, 14, 15, 16. Recent breakthroughs in combining the CRISPR library screens with droplet RNA-sequencing (RNA-seq) platforms demonstrated the capacity of monitoring the gene expression changes upon genetic perturbations in single cells (e.g., Perturb-seq, CRISP-seq, CROP-seq) 8, 9, 10, 11. However, the traditional CRISPR dropout/enrichment screens restricted the application to investigate functional elements associated with cell survival phenotypes. Furthermore, high-density CRISPR targeting of coding exons has been demonstrated to identify functional domains in genes 3, 4, 5, 6, 7. The integration of CRISPR (clustered, regularly interspaced, short palindromic repeats) with next-generation sequencing technology for high-throughput genetic screens is a powerful tool for discovering functional genes in various pathways and cellular contexts 1, 2. Distinct from other reported single-cell CRISPR screens focused on observing gene function and gene-to-gene/enhancer-to-gene regulation, sc-Tiling enables the capacity to identify regulatory mechanisms within a gene-coding region that dictate gene activity and therapeutic response. Here, we present “sc-Tiling,” which integrates a CRISPR gene-tiling screen with single-cell transcriptomic and protein structural analyses. Furthermore, breakthroughs in combining CRISPR library screens with single-cell droplet RNA sequencing (sc-RNAseq) platforms have revealed the capacity to monitor gene expression changes upon genetic perturbations at single-cell resolution. Recently, high-density mutagenesis via CRISPR tiling of gene-coding exons has been demonstrated to identify functional regions in genes. To date, CRISPR gene editing has primarily been applied to defining the role of individual genes. Identification of novel functional domains and characterization of detailed regulatory mechanisms in cancer-driving genes is critical for advanced cancer therapy.
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