CRISPR-based depletion method for resolving pseudogene contamination in Next Generation Sequencing (NGS) based genetic testing

ABSTRACT
Pseudogenes are non-functional or defective relics that are highly (>90%) homologous to their parental genes.
Even with approved guidelines for implementation, clinical tests based on next-generation sequencing (NGS)
are challenged because of the presence of these pseudogenes. High frequency of mutations in their DNA
sequence and high homology to parent genes prevents short NGS reads from mapping uniquely. Owing to this,
recent studies have estimated that one in seven pathogenic variants could not be detected by NGS assays.
Jumpcode Genomics proposes a CRISPR-based depletion technology to degrade molecules derived from
pseudogenes in NGS libraries. The removal of pseudogenes will both drastically reduce false-positive rates and
improve sensitivity and specificity in detecting clinically relevant SNPs. Jumpcode Genomics proposes to apply
its CRISPR-based depletion technology to remove pseudogene-derived molecules in library preparation
workflows for whole-genome sequencing (WGS) assays, specifically for newborn screening (NBS) clinical tests.
The goal is to increase the sensitivity and specificity of detecting disease-causing genetic mutations with high
accuracy. In this proposal, Jumpcode aims to extend this technology to a list of 80 genes in the NBS panel and
resolve mutations undetected in WGS by increasing the depletion of pseudogenes. Jumpcode performed a pilot
study by designing CRISPR-gRNAs (guide RNAs) targeted three genes in NBS panels demonstrated that in a
small experiment, the number of false-positive calls can be significantly reduced by carefully designing CRISPR-
gRNAs (guide RNAs) that target pseudogene molecules. This demonstrates not only the success of sequence-
specific depletion of pseudogenes but also the potential of enhancing variant calling accuracy in NGS panels.
This provides a basis for expanding to other genes in NBS lists in this proposal, as well as providing proof of
concept for applying DepleteX to other NGS applications in the future.
The specific aims of this Phase I proposal include computational design of CRISPR-gRNAs and optimization of
protocols to deplete pseudogenes, with the objective of resolving ambiguity in variant calling caused by
pseudogenes for a subset of genes in the NBS panel. Functional evaluation of the depletion assay will be
conducted, along with comparison with long-range PCR (LR-PCR) for variant calling. The assay will then be
independently evaluated in a clinical setting to ensure its accuracy and reliability. The successful demonstration
of this pseudogene depletion technology in Phase I will pave the way for future Phase II commercialization
efforts, including a clinical utility study to further validate its applicability in clinical settings. This innovative
approach holds great promise in improving the accuracy of genetic testing for newborn screening and other
clinical applications, ultimately enhancing patient outcomes and advancing precision medicine.

Grant Number: 1R43HG014080-01
NIH Institute/Center: NIH
Principal Investigator: Keith Brown