# CRISPR inhibition as an alternate for Cre-loxP

> **NIH NIH R21** · UNIV OF ARKANSAS FOR MED SCIS · 2020 · $199,980

## Abstract

PROJECT SUMMARY/ABSTRACT
Cre-loxP-based cell type-specific loss of function (LOF) studies have expanded our understanding
of skeletal biology and identified cellular and molecular mechanisms underpinning skeletal
pathologies. However, extensive use of the Cre-loxP system has revealed important
shortcomings of this methodology such as its activity in off-target cell types. Therefore, there is a
significant need for new methodologies to perform cell type-specific LOF studies. CRISPR
inhibition/interference (CRISPRi) is a derivative of the CRISPR-Cas9 system, developed to
suppress target genes, and constitutes an alternative method to perform LOF. In this
methodology, the two endonuclease domains of Cas9 protein are mutated to prevent Cas9's
ability to excise DNA. This dead Cas9 (dCas9) is fused to transcriptional suppressor domains,
such as KRAB, to produce the repressive fusion protein dCas9::KRAB. Once dCas9::KRAB
protein is directed to transcriptional start sites (TSS) of target genes via a single guide RNA
(sgRNA), this fusion protein suppresses expression of target genes. To test whether CRISPRi
can be utilized in vivo, we recently produced a murine model for global CRISPRi of Tnfsfs11.
Different founder lines of this murine model termed Tnfsf11gCRISPR exhibited dose-dependent
suppression of RANKL. Moreover, the phenotype of Tnfsf11gCRISPR founder lines that expressed
high levels of dCas9::KRAB was comparable to that of RANKL null mice. Based on the success
of gCRISPRi and the observed in vivo dose-dependency of CRISPRi, we hypothesize that
CRISPRi-based transgenes can be used to perform loss of function studies in mice that are more
specific than those performed with the Cre-loxP system but are as effective. This project will
produce osteocyte-specific CRISPRi models and compare the specificity and efficacy of cell type-
specific CRISPRi compared to the Cre-loxP system. Specifically, in the first aim we will test
whether a Dmp1 promoter driven CRISPRi transgene will suppress a target gene more
specifically than a Dmp1-Cre transgene. In the second aim, we will compare the efficacies of
CRISPRi and Cre-loxP systems. For this purpose, we will perform osteocyte-specific LOF of
RANKL using CRISPRi and Cre-loxP methodologies and compare the resultant phenotypes as
well as the decrease in RANKL levels. Establishing the specificity and effectiveness of in vivo
CRISPRi for cell type-specific LOF studies also lays the ground work for future studies aimed at
utilization of other advantages of the CRISPRi approach, such as suppression of multiple target
genes with one CRISPRi transgene.

## Key facts

- **NIH application ID:** 9877856
- **Project number:** 1R21AR076575-01
- **Recipient organization:** UNIV OF ARKANSAS FOR MED SCIS
- **Principal Investigator:** Melda Onal
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $199,980
- **Award type:** 1
- **Project period:** 2020-01-01 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9877856

## Citation

> US National Institutes of Health, RePORTER application 9877856, CRISPR inhibition as an alternate for Cre-loxP (1R21AR076575-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9877856. Licensed CC0.

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