Award Title: Transforming diagnostic certainty in developmental disorders using CRISPR-based saturation genome editing
Funder: The Academy of Medical Sciences

Abstract: Aims and objectives: We will apply saturation genome editing to the gene DDX3X to functionally classify all single nucleotide variants (SNV) and improve diagnostic certainty.

There is an urgent clinical need to improve the functional interpretation of genetic variants. This is required for the timely genetic diagnosis of developmental disorders. However, for most proteins, we do not know the functional consequence of the majority of genetic variants. This impairs the clinical interpretation of previously unobserved variants. Advances in CRISPR-Cas9 technologies allow us to specifically engineer all possible SNV in a gene at its endogenous locus. We propose to combine these technologies with in vitro massively parallel genetic screens to interrogate the functional significance of hundreds of genetic changes in a single experiment. This is known as Saturation Genome Editing (SGE).

The HAP1 cell line is near-haploid and derived from a chronic myeloid leukaemia line. Haploidy simplifies the design and interpretation of genetic screens. From publicly available data we have identified genes in which loss of function mutations cause developmental disorders and are essential in HAP1. We propose to apply SGE to these HAP1-essential genes in order to interrogate the functional consequence of every possible SNV. This grant will fund SGE of DDX3X, an X-linked RNA helicase of the DEAD-box protein family. Heterozygous loss of function mutations in DDX3X are the commonest genetic cause of intellectual disability in females. We will validate our results in a neural cell line, and by comparison with known clinical and population genetic variation.

Lay Abstract: Genes act as a code which is ‘read’ to make proteins, the building blocks of cells. In 2020, we still do not properly understand our genetic code. We all carry genetic changes, but most do not cause problems. However, in a genetic condition, a change in the genetic code changes the function of a protein and alters a child’s development. We check a person’s genetic code when we suspect that they have a genetic condition. Unfortunately, we cannot always predict which genetic changes will alter a protein’s function. Therefore, it can be difficult to decide whether a child’s difficulties are due to the genetic changes that we have observed. This often makes diagnosis difficult. This is particularly true in young children or unborn babies, as the true extent of their difficulties may not yet be fully known. However, diagnosing children when they are young can improve treatment and reduce the disability caused by a genetic condition. Families tell us that earlier and quicker diagnosis would be useful to them. Our experiment aims to understand better which genetic changes will cause a child difficulties.

In our experiment we will artificially change the genetic code in human cells grown in a dish. We will test thousands of changes to identify which ones prevent proteins from working properly. This will help us to understand the impact that these genetic changes could have in a child. This will help doctors to interpret genetic tests and diagnose children earlier.

Research Activity Codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health Categories: CONGENITAL DISORDERS