Multiplexed Functional Assessment of Genetic Variants in CARD11

Abstract

Summary Genetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a “cloning-free” saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.

Saturation variant interpretation using CRISPR prime editing

“…While each library had a different pegRNA architecture and targeted a different area of NPC1 , we noted several patterns in the editing outcomes with SPE across the four regions. Similar to previous SGE studies using homology-directed repair 8 , the universal silent PAM-destroying mutation with no secondary mutation was disproportionately the most common editing outcome (Supplementary Figure S4). It has been documented by others that PAM-modifying mutations tend to occur at a greater frequency than other edits made with the same pegRNA in prime editing 11,32 .…”

“…These assays, however, often uncouple the variants-of-interest from their native genomic context being overexpressed as cDNAs from artificial cassettes or in non-human cell types 4,5 . This has been addressed by saturation genome editing (SGE), which uses CRISPR-Cas9 genome editing to introduce all possible SNVs into a targeted region of the endogenous genomic locus 6–8 .…”

“…In contrast to HDR, prime editing does not require exogenous donor templates and provides predictable editing outcomes and high product purity. In addition, though SGE has been recently expanded to the diploid TMD8 cell line 8 , previous studies have relied on the near haploid HAP1 cell line 6,7,12 . While HAP1 cells simplify interpretation by allowing for functional analysis of variants in isolation, they limit SGE to genes expressed or phenotypes elicitable in these cells.…”

Variant Library Annotation Tool (VaLiAnT): an oligonucleotide library design and annotation tool for Saturation Genome Editing and other Deep Mutational Scanning experiments

“…The reverse-complement option in VaLiAnT permits the generation of variant sequences for strand-specific applications. For example, SGE using single-stranded oligo donor (ssODN) has been reported to streamline experimental procedures, however strand-specific differences in HDR-rate have been observed with ssODN template and orientation should be chosen depending on editing context (Kan, Ruis, Takasugi, & Hendrickson, 2017; Meitlis et al, 2020). Therefore, VaLiAnT could be used to generate strand-specific libraries for ssODN-based SGE experiments.…”

“…However, the rate of discovery of new variants in disease loci calls for a proactive approach to functional assessment using high-throughput, Multiplexed Assays of Variant Effect (MAVEs) and Massively Parallel Reporter Assays (MPRAs) for coding and noncoding loci, respectively (Starita et al, 2017). A range of MAVE and MPRA technologies have been developed to produce variant effect maps, including yeast complementation (Hietpas, Jensen, & Bolon, 2011;Starr et al, 2020;Sun et al, 2016), cell display assays (Forsyth et al, 2013;Starr et al, 2020), FACs-based screens (Matreyek et al, 2018) including sort-Seq MPRA (Kinney, Murugan, Callan, & Cox, 2010), RNA-Seq MPRA (Melnikov et al, 2012) and CRISPR/Cas9-based saturation genome editing (SGE) in human cells (Findlay et al, 2018;Meitlis et al, 2020). A distinct benefit of SGE is that variants are assessed within the endogenous genomic context, allowing interrogation of complex mutational consequence, including splicing effects.…”