High-throughput phenotypic screening for functionally characterizing alt. exons

Division Of Basic Sciences - Nci

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Abstract

Aim A In recent work performed at NCI/NIH, we have applied molecular engineering strategies to further boost the editing efficiency of CHyMErA. We have shown that addition of 6 nuclear localization signals at the C-terminus of Cas12a stimulates the editing efficiency of CHyMErA by promoting nuclear accumulation and stabilization of Cas12a nuclease. We are currently applying the improved CHyMErA screening platform to identify alternative exons that impact on cell fitness and proliferation in cancer cells. Comprehensive deletion of frame-preserving alternative exons will be performed using the CHyMErA screening platform across a panel of 7 diverse human cancer cell lines (A375, HCT116, MDA-MB-231, A549, Jurkat, K562, HAP1). These screens will enable the systematic cataloguing of the core and context-specific alternative fitness exons, along with providing insights into the functional complexity of the transcriptome. Aim B We have previously developed CHyMErA, a powerful and versatile tool for combinatorial perturbation of genomic loci, which is based on the expression of hybrid Cas9 and Cas12a gRNAs. This tool has been successfully applied for dropout and enrichment cell fitness screens. Compared to conventional single-cell transcriptomics, CRISPR screens coupled to scRNA-Seq require the capture of non-polyadenylated gRNAs in addition to the capture of mRNA in order to assign perturbations to consequential transcriptomic outputs. CHyMErA, in its current form, does not allow capturing of guide sequences using any scRNA-Seq platform. Therefore, we aim to transform CHyMErA to a perturbation tool that is amenable to capturing both the mRNA transcriptome and gRNAs at the single-cell level. To achieve this, we will introduce capture sequences in the middle of the tracrRNA of Cas9 and at the end of Cas12 gRNAs. These capture sequences will be recognized by customized RT barcoded primers during the first step of generating scRNA-Seq libraries. In parallel, barcoded polyT RT primers will be used to capture polyadenylated mRNA. Capture sequences have recently been used for a Cas9-based CRISPR platform and made available for use with the 3 Direct Capture 10x Genomics platform. We aim to use these capture sequences in our system but need to ensure that their incorporation does not impact the editing efficiency of CHyMErA. To test the suitability of scCHyMErA for combinatorial CRISPR perturbations coupled to scRNA-Seq, we will generate a small library targeting 450 genes using dual-targeting hgRNAs directing both Cas9 and the Cas12a to target the same gene. Two independent hgRNAs will be designed per gene resulting in 1,000 library elements (including controls) that will be cloned as a pool into a lentiviral backbone following protocols that we have mastered in the past. For our first screen we will focus on targeting genes encoding RNA binding proteins (RBPs) which regulate RNA processing events including alternative splicing and polyadenylation. These processes underlie important cell fate decisions and their disruption is linked to numerous diseases including hematological malignancies. The hgRNA library will be transduced into embryonic stem cells and subsequently subjected to single-cell RNA-Seq analysis profiling using standard 10x Genomics protocols.The transcriptomes will be sequenced on a NovaSeq 6000 S2 flow cell resulting in 4 billion reads equal to 50k reads per profiled cell and data will be analyzed using established computational pipelines. These data will not only establish scCHyMErA-Seq as a tool for CRISPR perturbation with scRNA-Seq readout but also may uncover genes and RNA-related pathways that play an important role in stem cell maintenance and pluripotency. Future screens will be focused on targeting tissue-regulated alternative cassette exons with scCHyMErA-Seq in order to uncover the postranscriptional splicing events that underlie developmental trajectories.