Abstract

Somatic and germline genetic mutations can lead to several health conditions, including cancers and developmental disorders. These genetic variants could be identified in individuals through genomic sequencing. However, it remains challenging to predict the pathogenicity of most identified variants due to insufficient or conflicting evidence of their effect. This group of variants was clinically classified as variants of uncertain significance (VUS). The VUS could be resolved by accumulating clinical evidence and sequencing data. Nevertheless, this conventional strategy might be too slow to be reliably useful for diagnostic purposes. New diagnostic tools are urgently needed for accurate diagnosis and prognostication for patients. Saturation genome editing (SGE) utilizes CRISPR-Cas9 technologies to engineer thousands of genetic variants into the endogenous genetic locus of a pool of cells and directly test their functional impact in vitro. We have applied SGE to several exons of cancer-related and neurodevelopmental-disorder-related genes. In particular, we have completed a functional map for every coding nucleotide of DDX3X. Heterozygous loss-of-function mutations in DDX3X are the commonest genetic cause of female intellectual disability. We also showed that SGE has advantages in identifying deleterious intronic and splicing variants, which other saturation-mutagenesis-based approaches cannot achieve.