PROJECT SUMMARY/ABSTRACT
SCD is a heritable disease, which affects a patient's red blood cells (RBCs). This monogenic disorder is
caused by a single nucleotide polymorphism (SNP) within the HBB gene. Despite progress in the treatment
of SCD regarding early screenings, prevention of infections, and blood transfusions, the life expectancy for
SCD patients is still reduced by about 30 years. Currently, allogeneic hematopoietic stem cell
transplantation (HSCT) is the only curative treatment available. Unfortunately, the process is invasive and
associated with high risk of graft-versus-host-disease, infection, and infertility. CRISPR-based gene editing
is a powerful therapeutic tool for potentially curing a wide variety of diseases. However, low editing
efficiency can result in unedited HSPCs outcompeting edited ones, resulting in diminished therapeutic
impact. Current methods for maximizing the percentage of edited cells rely on GFP or surface protein
sequences to be contained within the homologous donor DNA, complex optical assays and cell sorting to
establish cell populations with >85% editing efficiency. We propose to develop a versatile and easy-to-use
platform to monitor and optimize the editing efficiency of CRISPR/Cas9 for SCD gene therapy
applications. This in vitro platform utilizes multiplex CRISPR-transistors to quantify the amount of a
specific sequence within an unamplified genomic DNA sample without the bias associated with the artifacts
of library preparation like other sequencing-based methods. The electronic platform provides rapid readout
with low sample input requirement. By combining the programmability of RNA-guided CRISPR-Cas
technology with the scalability of nano-electronics, the proposed project provides a flexible, and simple to
use ex-vivo monitoring solution for a comprehensive and effective gene therapy quality control. We
will expand CRISPR-transistor design in Aim 1 to yield a sensor which employs a variety of gRNA designs
and RNA-guided Cas nucleases to electronically detect and quantify single nucleotide changes using SCD
as a genetic model. In Aim 2, we will scale up this technology design and fabricate a multiplex gFET
capable of analyzing a single sample with up to 16 different RNA-guided Cas complexes simultaneously
without amplification. In Aim 3, we will utilize this multi-plex CRISPR-transistor platform to rapidly assess
the ex-vivo CRISPR/Cas9 HBB editing efficiency of HSPCs from patients with SCD. In addition, we will
leverage the flexibility of CRISPR-transistor to establish an ON/OFF-target evaluation of the RNA-guided
Cas nuclease in the presence of chromatin structures and compare against existing technologies for off-
target screening, like CIRCLE-seq and genome wide. This project will demonstrate a facile, general
platform for quantification of editing efficiency that has the potential to shorten the processing time,
reducing sample and complexity necessary to ensure high quality of ex-vivo gene therapy.
