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B - Gene Targeting and Gene Correction -> B1 – Gene Targeting and Gene Correction – In Vivo Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

1210: Controlling CRISPR-Cas9 Genome Editing in Human Cells Using a Small-Molecule Inducible Degradation Strategy

Type: Poster Session

Poster Board Number: 1210
Presentation Details
Session Title: Thursday Poster Session
Start Time: 5/18/2023 12:00
End Time: 5/18/2023 14:00

INTRODUCTION: CRISPR/Cas9 offers the advantage of targeting and cutting specific genes of interest with high precision. However, prolonged Cas9 activity has negative consequences such as off-target editing, genotoxicity, immunogenicity, and undesired on-target modifications. Current methods to control the CRISPR/Cas9 system include various small molecules, inhibitors, light and magnetic fields; however, these methods lack high-resolution temporal control over Cas9 activity in vivo and may leave behind residual active Cas9. Recent work has shown that a degron can be controlled by FDA-approved pomalidomide (POM). Here, we attach degrons to Cas9 and characterize the effectiveness of the degrons in degrading Cas9 and modifying the on- and off-target editing efficiency with POM.
METHODS: Cas9-degron was inserted into human embryonic stem (H9s) and kidney (HEK293T) cells via lentiviral transduction. Varying amounts of POM were added to determine the optimal amount of drug that does not suppress cell growth or induce apoptosis. Studies were conducted to determine Cas9 protein levels once POM is added. To confirm that the degron does not hinder on-target editing efficiency, the AAVS1 Site 10 locus was targeted. DNA was extracted after two days, followed by PCR and Next Generation Sequencing. Replicates were lysed and Western Blotting was used to quantify protein. The RGEN and CRISPAltRations tool helped characterize on- and off-target editing.
RESULTS: With the addition of POM, there is an overall decrease in Cas9-deg levels as shown by Western Blotting (Figure A). Withdrawing POM shows that Cas9 levels return within 24 hours, while a single dosage of POM can continuously degrade Cas9 protein up to 96 hours (Figure B). On-target editing efficiency (measured by indel frequency %) of wild-type (WT) Cas9 and Cas9-deg was similar (Figure C)—confirming that Cas9 activity is not hindered by the degron units. Adding POM to decrease Cas9 levels affected on-target editing efficiency significantly suggesting that Cas9-degron off-target editing activity will also decrease.
CONCLUSION: The small molecule, POM, reversibly controls degron-engineered Cas9 protein levels as well as gene editing activity within human cells. Since, this small molecule inducer has been considered safe and is known to pass the blood-brain barrier, the system tested here has strong potential for reversible control of genome editing in vivo in many tissues, including the central nervous system.

Namita Khajanchi1, Vrusha Chirayu Patel1, Krishanu Saha2

1Biomedical Engineering, University of Wisconsin -- Madison, Madison, WI,2University of Wisconsin-Madison, Madison, WI
 N. Khajanchi: None.

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