Guide RNA reprogramming facilitates minimized tracrRNA-dependent off-target and versatile CRISPR/Cas9 engineering
Year: 2026
Type: article
Abstract: While innovative, current CRISPR-Cas9 systems face safety concerns and practical hurdles, notably sequence-independent, noncanonical off-targeting. We demonstrate that the crRNA:tracrRNA duplex in guide RNAs (gRNA) is both splittable and reprogrammable. This property, however, enables endogenous RNAs with crRNA-like sequences to hijack any gRNAs, causing low-frequency yet pervasive tracrRNA-dependent off-target (TDO) effects. Using machine learning trained on high-throughput gRNA variant screens, we derive optimal gRNA-designing rules and engineer crRNA variants mismatched to the human/mouse transcriptomes, thereby minimizing TDO. By leveraging splittability and reprogrammability, we develop reprogrammable tracrRNAs for CRISPRa-based mRNA detection and redesign scaffolds to curb PAM-less Cas9-mediated “self-editing”. We further create a separately expressed gRNA (segRNA) platform featuring split tracrRNAs and non-repetitive tandem crRNAs, enabling multiplexed editing of up to six genes and functional enhancer annotation in stem cells. Our findings uncover a previously overlooked off-target mechanism and offer versatile strategies to enhance the safety and utility of CRISPR systems. Leveraging the reprogrammability and splittability of CRISPR gRNAs, the authors find that endogenous crRNA-like RNAs hijack tracrRNAs to cause low-frequency yet pervasive tracrRNA-dependent off-target (TDO) effects. This same principle also offers versatile strategies to enhance CRISPR safety and utility.
Source: Nature Communications
Institutions: Shanghai Jiao Tong University, Shanghai Ninth People's Hospital, Shandong University, Qilu Hospital of Shandong University, ShanghaiTech University, International Peace Maternity & Child Health Hospital, Chinese Academy of Sciences, Center for Excellence in Molecular Cell Science, University of Chinese Academy of Sciences, Nanjing Medical University, Renji Hospital, First Affiliated Hospital Zhejiang University, Zhejiang University, Fudan University, Children's Hospital of Fudan University, State Key Laboratory of Medical Neurobiology, Frontiers Center for Brain Science of the Ministry of Education
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $6,990
OpenAlex ID: W7165045066