Cas9 Degradation in Human Cells Using Phage Anti-CRISPR Proteins
Zuriah Meacham, Luisa Arake de Tacca, Joseph Bondy-Denomy, David Rabuka, Michael Schelle
Abstract
Bacteriophages encode anti-CRISPR (Acr) proteins that inactivate CRISPR-Cas bacterial immune systems, allowing successful invasion, replication, and prophage integration. Acr proteins inhibit CRISPR-Cas systems using a wide variety of mechanisms. AcrIIA1 is encoded by numerous phages and plasmids, binds specifically to the Cas9 HNH domain, and was the first Acr discovered to inhibit SpyCas9. Here, we report the observation of AcrIIA1-induced degradation of SpyCas9 and SauCas9 in human cell culture, the first example of Acr-induced degradation of CRISPR-Cas nucleases in human cells. AcrIIA1-induced degradation of SpyCas9 is abolished by mutations in AcrIIA1 that break a direct physical interaction between the 2 proteins. Targeted Cas9 protein degradation by AcrIIA1 could modulate Cas9 nuclease activity in human therapies. The small size and specificity of AcrIIA1 could be used in a CRISPR-Cas proteolysis-targeting chimera (PROTAC), providing a tool for developing safe and precise gene editing applications.
Introduction
CRISPR (clustered regularly interspaced short palindromic repeats) arrays contain fragments of DNA that bacteria use as defense against invading nucleic acids [1,2]. RNA-guided CRISPR-associated (Cas) nucleases identify invaders by first binding to a short protospacer adjacent motif (PAM) and then through Watson–Crick base-pairing, which leads to nucleic acid cleavage [3]. Phages have evolved CRISPR inhibitors that aid in evasion of the CRISPR defense and enhance the transmission of mobile genetic elements (MGEs) [4]. Anti-CRISPR (Acr) proteins inactivate the CRISPR-Cas immune system of bacteria [4–7]. The first example of phage-encoded Acr proteins were found to inhibit the Class 1 Type I CRISPR-Cas systems [8,9]. Shortly after this discovery, the first antagonists of Class 2 Type II CRISPR-Cas systems, including the clinically relevant SpyCas9, were identified in Listeria prophages [10,11]. AcrIIA1 was revealed to be widespread across Firmicutes prophages and MGEs and has even been used as a marker for the discovery of new Acr proteins [10]. AcrIIA1 is a broad-spectrum Cas9 inhibitor, capable of inhibiting multiple Cas9 orthologs [12]. AcrIIA1 inhibits multiple Type II-C Cas9 enzymes as well as the more common and therapeutically relevant Type II-A nucleases, including SauCas9 and SpyCas9 [12].
Materials and methods
Plasmid cloning
Three plasmids containing AcrIIA1 were constructed expressing either: a native bacterial codon acrIIA1 (AcrIIA1-bac), a human codon-optimized acrIIA1 (AcrIIA1-hum), or AcrIIA1-hum with an HA-tag on the N-terminus (AcrIIA1-HA). These were ordered as gene fragments from Twist Bioscience and cloned into Twist’s CMV expression vector using HindIII and BamHI restriction sites. AcrVA1 and AcrIIA4 used as controls were codon-optimized for human expression and ordered and cloned exactly as AcrIIA1. The SpyCas9 plasmid was purchased from Genscript with BbsI cloning sites for guide addition. An HBB guide was added to the SpyCas9 plasmid through the oligo anneal protocol provided from Dr. Feng Zhang’s lab available online under “PX330 cloning protocol.” The oligos used to make the HBB target are listed in S1 File.
Results and discussion
AcrIIA1 inhibits Cas9 gene editing in human cells
We transfected HEK293T human cells with a plasmid expressing SpyCas9 and a guide targeting the hemoglobin beta (HBB) locus and a second plasmid expressing AcrIIA1 (Fig 1A). Similar to previous results [10], AcrIIA1 encoded with native bacterial codons (AcrIIA1-bac) does not fully inhibit SpyCas9 editing. However, expression of a human codon optimized version of the acrIIA1 gene (AcrIIA1-hum) fully inhibited SpyCas9 editing. Editing at a known HBB off-target site (HBD) was also fully inhibited. Titration of AcrIIA1-bac plasmid showed a dose-dependent increase in SpyCas9 editing (Fig 1B). Western blot analysis shows a concomitant increase in AcrIIA1 expression with increasing plasmid amount (Fig 2B). The AcrIIA1-hum construct was able to inhibit SpyCas9 editing at 0.5:1 plasmid weight:weight ratio. We also assessed AcrIIA1 inhibition of SpyCas9 in a second human cell line (Hep G2 liver cells) and observed a similar reduction in editing activity (S1 Fig).
Discussion
In this work, we show for the first time that an anti-CRISPR protein is capable of inducing the degradation of a CRISPR-Cas nuclease in human cells. Destabilization or degradation of a Cas protein by an Acr is an uncommon mechanism. AcrIIA1 was previously shown to inhibit and induce degradation of Cas9 orthologs in Listeria [12]. Key binding residues were elucidated on both the Acr and Cas9 protein, explaining the broad phylogenetic distribution of the AcrIIA1 family and breadth of Cas9 inhibition. While the exact mechanism of AcrIIA1-induced Cas9 degradation remains unknown, the authors concluded that the degradation mechanism was likely to be limited to certain bacterial species where Cas9 and AcrIIA1 are naturally found. In this report, we show that AcrIIA1 induces degradation of SpyCas9 and SauCas9 by direct binding in human cells. This surprising observation could be used to develop a Cas9 PROTAC, which is capable of controlled Cas9 degradation, similar to previously engineered auxin inducible degron fusions [15].
Acknowledgments
We want to thank the members of Acrigen Biosciences for their assistance and helpful discussion.
Citation: Meacham Z, de Tacca LA, Bondy-Denomy J, Rabuka D, Schelle M (2023) Cas9 degradation in human cells using phage anti-CRISPR proteins. PLoS Biol 21(12): e3002431. https://doi.org/10.1371/journal.pbio.3002431
Academic Editor: Franklin L. Nobrega, University of Southampton, UNITED KINGDOM
Received: March 23, 2023; Accepted: November 14, 2023; Published: December 8, 2023
Copyright: © 2023 Meacham et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the National Institute of General Medical Sciences (R43GM145002) to DR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: D.R. and J.B.D. are founders of Acrigen Biosciences. D.R., M.S., Z.M., and L.T. are employees of Acrigen Biosciences.
Abbreviations: CRISPR, clustered regularly interspaced short palindromic repeats; MGE, mobile genetic element; PAM, protospacer adjacent motif; PROTAC, proteolysis-targeting chimera
https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002431&rev=2#ack
