Wednesday, April 2, 2025 4/2/2025

CRISPR-based transistors for high throughput multiplexed monitoring of CRISPR-based editing efficiency for Sickle cell disease

Award Number: R01HL161361
ORGANIZATION: NATIONAL HEART, LUNG, & BLOOD INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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CRISPR-based transistors for high throughput multiplexed monitoring of CRISPR-based editing efficiency for Sickle cell disease - 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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $388,206 )
2025	2025	UNIVERSITY OF CALIFORNIA, SAN DIEGO	9500 GILMAN DR	LA JOLLA	CA	92093	SAN DIEGO	USA	Blood Diseases and Resources Research	000	4	3/3/2025	NON-COMPETING CONTINUATION	$388,206
														Subtotal = $388,206

Issue Date FY: 2024 ( Subtotal = $422,713 )
2024	2024	UNIVERSITY OF CALIFORNIA, SAN DIEGO	9500 GILMAN DR	LA JOLLA	CA	92093	SAN DIEGO	USA	Blood Diseases and Resources Research	001	3	5/20/2024	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$422,713
2024	2023	KECK GRADUATE INSTITUTE OF APPLIED LIFE SCIENCES	535 WATSON DR	CLAREMONT	CA	91711	LOS ANGELES	USA	Blood Diseases and Resources Research	000	2	5/16/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $422,713

Issue Date FY: 2023 ( Subtotal = $409,227 )
2023	2023	KECK GRADUATE INSTITUTE OF APPLIED LIFE SCIENCES	535 WATSON DR	CLAREMONT	CA	91711	LOS ANGELES	USA	Blood Diseases and Resources Research	000	2	12/22/2022	NON-COMPETING CONTINUATION	$368,304
2023	2023	KECK GRADUATE INSTITUTE OF APPLIED LIFE SCIENCES	535 WATSON DR	CLAREMONT	CA	91711	LOS ANGELES	USA	Blood Diseases and Resources Research	001	2	4/4/2023	NON-COMPETING CONTINUATION	$40,923
														Subtotal = $409,227

Issue Date FY: 2022 ( Subtotal = $405,375 )
2022	2022	KECK GRADUATE INSTITUTE OF APPLIED LIFE SCIENCES	535 WATSON DR	CLAREMONT	CA	91711	LOS ANGELES	USA	Blood Diseases and Resources Research	000	1	1/7/2022	NEW	$405,375
														Subtotal = $405,375

Grand Total All Awards = $1,625,521

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CRISPR-based transistors for high throughput multiplexed monitoring of CRISPR-based editing efficiency for Sickle cell disease

Award Number: R01HL161361

ORGANIZATION: NATIONAL HEART, LUNG, & BLOOD INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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