Matreyek, K. A. | bioRxiv Science

bioRxiv2018

On the design of CRISPR-based single cell molecular screens

Several groups recently reported coupling CRISPR/Cas9 perturbations and single cell RNA-seq as a potentially powerful approach for forward genetics. Here we demonstrate that vector designs for such screens that rely on cis linkage of guides and distally located barcodes suffer from swapping of intended guide-barcode associations at rates approaching 50% due to template switching during lentivirus production, greatly reducing sensitivity. We optimize a published strategy, CROP-seq, that instead uses a Pol II transcribed copy of the sgRNA sequence itself, doubling the rate at which guides are assigned to cells to 94%. We confirm this strategy performs robustly and further explore experimental best practices for CRISPR/Cas9-based single cell molecular screens.

genomics↗

bioRxiv2018

Multiplex Assessment of Protein Variant Abundance by Massively Parallel Sequencing

Determining the pathogenicity of human genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes will likely require generalizable, scalable assays. Here we describe Variant Abundance by Massively Parallel Sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance in a single experiment. We apply VAMP-seq to quantify the abundance of 7,595 single amino acid variants of two proteins, PTEN and TPMT, in which functional variants are clinically actionable. We identify 1,079 PTEN and 805 TPMT single amino acid variants that result in low protein abundance, and may be pathogenic or alter drug metabolism, respectively. We observe selection for low-abundance PTEN variants in cancer, and our abundance data suggest that a PTEN variant accounting for ~10% of PTEN missense variants in melanomas functions via a dominant negative mechanism. Finally, we demonstrate that VAMP-seq can be applied to other genes, highlighting its potential as a generalizable assay for characterizing missense variants.

genetics↗