# Discovery and engineering of CRISPR/Cas systems

> **NIH NIH R35** · UNIVERSITY OF FLORIDA · 2024 · $366,921

## Abstract

PROJECT SUMMARY/ABSTRACT:
The long-term objectives of the proposed program are to (i) discover type V CRISPR/Cas systems in exotic
microorganisms with unique features, (ii) elucidate a deeper understanding of the rules and mechanisms of
CRISPR/Cas and apply it for engineering and improving its activity, and (iii) apply them for gene editing and
diagnostic applications for a range of diseases. Although the type II CRISPR/Cas9 is the most studied genome
editing tool, the type V CRISPR/Cas12 systems are the most diverse with a wide range of functionally distinct
single-effector Cas12a-k nucleases that are emerging as next-generation tools for both genome editing and
nucleic acid detection. The central hypothesis is that (i) since the type V systems are most diverse and relatively
newer, only a handful (<5%) of these systems have been properly studied, while a vast majority of these systems
are understudied and poorly characterized and therefore, a systematic study of these systems will enable novel
tools for genome engineering, chromatin imaging, base editing, and diagnostics. (ii) A deeper understanding of
the sequence-structure-activity relationship by engineering crRNA and Cas will enable the development of
improved tools for metagenomic analysis, combinatorial enzymology, and multiplexing strategies for genome
editing and diagnostic applications. While the type V CRISPR/Cas share challenges of poor delivery, low gene
correction efficiency, and high off-target cleavage associated with other CRISPR-based genome editing tools,
they possess both orthogonal and overlapping challenges for diagnostic applications, including a) low catalytic
efficiency or poor sensitivity, b) high tolerance of mismatches or low specificity, c) poor stability for deployment,
and d) lack of control, desirable for multiplexing. In the first program, novel orthologs of type V CRISPR/Cas
systems will be discovered by metagenomic mining of exotic microorganisms that can thrive at extreme
conditions followed by expression and purification of Cas enzymes and crRNAs, identification of protospacer
adjacent motif requirement, and testing of enzymatic activity in a high-throughput fashion. In the second
program, crRNAs and Cas proteins will be modified with various strategies to improve target specificity and
activity. Modified crRNAs and Cas would allow elucidation of mechanisms of CRISPR/Cas systems that could
further allow improved detection of target DNA or RNA. Finally, integrating novel and engineered CRISPR/Cas
with model systems would enable the development of multiplexed technologies that will have broader impacts
in the detection and treatment of a wide range of diseases. The PI's lab has already made significant
contributions in all three proposed programs with several key collaborations and publications and is poised
to run a successful research and training program. The expected outcomes of the support from the Maximizing
Investigators' Research Award (MIRA) for Early ...

## Key facts

- **NIH application ID:** 10908290
- **Project number:** 5R35GM147788-03
- **Recipient organization:** UNIVERSITY OF FLORIDA
- **Principal Investigator:** Piyush K Jain
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $366,921
- **Award type:** 5
- **Project period:** 2022-09-01 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10908290

## Citation

> US National Institutes of Health, RePORTER application 10908290, Discovery and engineering of CRISPR/Cas systems (5R35GM147788-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10908290. Licensed CC0.

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