# A Molecular Grammar for Guide RNAs (gRNAs) with Engineered Secondary Structures

> **NIH NIH R21** · UNIVERSITY OF NORTH CAROLINA GREENSBORO · 2022 · $181,875

## Abstract

CRISPR effectors like Cas9 and Cas12a have emerged as powerful tools in biomedical research for their ability
to introduce targeted mutations in living cells and, consequentially, for this ability they hold significant therapeutic
potential for treating genetic disorders—despite also carrying significant clinical risk that they may introduce ‘off-
target’ or unintended mutations. While the power of CRISPR effectors lies in the fact that the sequences they
recognize and target are complementary to a modular, ‘programmable’ segment of their RNA cofactors (their
‘guide RNAs’ or gRNAs), their mutational activity can be triggered at nucleotide sequences with imperfect
complementarity to their gRNAs as well, unpredictably. Obviously, the possibility of uncontrolled mutation raises
red flags for both patients and clinicians and so far, CRISPR gene therapies have been focused on highly
specialized genetic situations. Further improvements to CRISPR specificity are necessary, not only to mitigate
clinical risk, but also to drive new applications of CRISPR—for example, if single nucleotide variants (SNVs)
could be reliably discriminated, it would allow for allele-specific gene editing of autosomal dominant disorders,
where often we would need to discriminate between small sequence variations between the ‘healthy’ and
‘disease’ alleles but which current CRISPR technologies cannot consistently do. We recently demonstrated the
feasibility of an approach that is capable of improving CRISPR effector specificity by orders-of-magnitude, and
in such a way that it can be synergistically applied to many of the other previously-developed techniques to
improve specificity further. By adding extra nucleotides to the gRNA (x-gRNA) and designing the extended
sequence to form ‘hairpin’ secondary structures with the DNA-targeting segment of the gRNA (hairpin-gRNAs or
hp-gRNAs) that destabilize interactions with off-targets, we could generate x/hp-gRNAs that significantly limited
off-target activity while maintaining on-target mutational activity in CRISPR effector variants derived from four
different organisms and one engineered derivative. The long-term goal is therefore to understand the rules for
designing extended sequences in x-gRNAs that would result in ultra-specificity for divergent CRISPR effectors
at any CRISPR-targetable site. To achieve that goal, in this R21 we will perform an exhaustive screen of
randomized x-gRNA libraries targeting different clinically-relevant sites and identify what common sequence
and/or secondary-structure features of those x-gRNAs drive significant increases in specificity. While the
riskiness of this proposal is that there may not be “universal design rules”, per se, for all x/hp-gRNA designs
and targets, this work will nevertheless provide a practical (design-free) platform for researchers to empirically
generate ultra-specific x-gRNAs for any target of interest for any CRISPR effector. The likely reward is that
synergistic use of x-g...

## Key facts

- **NIH application ID:** 10511156
- **Project number:** 1R21EB033595-01
- **Recipient organization:** UNIVERSITY OF NORTH CAROLINA GREENSBORO
- **Principal Investigator:** Eric Alan Josephs
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $181,875
- **Award type:** 1
- **Project period:** 2022-08-15 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10511156

## Citation

> US National Institutes of Health, RePORTER application 10511156, A Molecular Grammar for Guide RNAs (gRNAs) with Engineered Secondary Structures (1R21EB033595-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10511156. Licensed CC0.

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