TY - JOUR
T1 - Engineered Cas9 variants bypass Keap1-mediated degradation in human cells and enhance epigenome editing efficiency
AU - Chen, Jianfeng
AU - Su, Siyuan
AU - Pickar-Oliver, Adrian
AU - Chiarella, Anna M.
AU - Hahn, Quentin
AU - Goldfarb, Dennis
AU - Cloer, Erica W.
AU - Small, George W.
AU - Sivashankar, Smaran
AU - Ramsden, Dale A.
AU - Major, Michael B.
AU - Hathaway, Nathaniel A.
AU - Gersbach, Charles A.
AU - Liu, Pengda
N1 - Publisher Copyright:
© The Author(s) 2024.
PY - 2024/10/28
Y1 - 2024/10/28
N2 - As a potent and convenient genome-editing tool, Cas9 has been widely used in biomedical research and evaluated in treating human diseases. Numerous engineered variants of Cas9, dCas9 and other related prokaryotic endonucleases have been identified. However, as these bacterial enzymes are not naturally present in mammalian cells, whether and how bacterial Cas9 proteins are recognized and regulated by mammalian hosts remain poorly understood. Here, we identify Keap1 as a mammalian endogenous E3 ligase that targets Cas9/dCas9/Fanzor for ubiquitination and degradation in an ‘ETGE’-like degron-dependent manner. Cas9-‘ETGE’-like degron mutants evading Keap1 recognition display enhanced gene editing ability in cells. dCas9-‘ETGE’-like degron mutants exert extended protein half-life and protein retention on chromatin, leading to improved CRISPRa and CRISPRi efficacy. Moreover, Cas9 binding to Keap1 also impairs Keap1 function by competing with Keap1 substrates or binding partners for Keap1 binding, while engineered Cas9 mutants show less perturbation of Keap1 biology. Thus, our study reveals a mammalian specific Cas9 regulation and provides new Cas9 designs not only with enhanced gene regulatory capacity but also with minimal effects on disrupting endogenous Keap1 signaling.
AB - As a potent and convenient genome-editing tool, Cas9 has been widely used in biomedical research and evaluated in treating human diseases. Numerous engineered variants of Cas9, dCas9 and other related prokaryotic endonucleases have been identified. However, as these bacterial enzymes are not naturally present in mammalian cells, whether and how bacterial Cas9 proteins are recognized and regulated by mammalian hosts remain poorly understood. Here, we identify Keap1 as a mammalian endogenous E3 ligase that targets Cas9/dCas9/Fanzor for ubiquitination and degradation in an ‘ETGE’-like degron-dependent manner. Cas9-‘ETGE’-like degron mutants evading Keap1 recognition display enhanced gene editing ability in cells. dCas9-‘ETGE’-like degron mutants exert extended protein half-life and protein retention on chromatin, leading to improved CRISPRa and CRISPRi efficacy. Moreover, Cas9 binding to Keap1 also impairs Keap1 function by competing with Keap1 substrates or binding partners for Keap1 binding, while engineered Cas9 mutants show less perturbation of Keap1 biology. Thus, our study reveals a mammalian specific Cas9 regulation and provides new Cas9 designs not only with enhanced gene regulatory capacity but also with minimal effects on disrupting endogenous Keap1 signaling.
UR - http://www.scopus.com/inward/record.url?scp=85208160745&partnerID=8YFLogxK
U2 - 10.1093/nar/gkae761
DO - 10.1093/nar/gkae761
M3 - Article
C2 - 39228373
AN - SCOPUS:85208160745
SN - 0305-1048
VL - 52
SP - 11536
EP - 11551
JO - Nucleic acids research
JF - Nucleic acids research
IS - 19
ER -