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Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

Title
Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes
Authors
Kim, BoilKim, Ji HoonChun, MinjeongPark, InahKwak, DamhyeonChoi, MijungKim, KyungjinChoe, Han Kyoung
DGIST Authors
Kim, Boil; Kim, Ji Hoon; Chun, Minjeong; Park, Inah; Kwak, Damhyeon; Choi, Mijung; Kim, Kyungjin; Choe, Han Kyoung
Issue Date
2021-01
Citation
Scientific Reports, 11(1), 2575
Type
Article
ISSN
2045-2322
Abstract
The mammalian molecular clock is based on a transcription-translation feedback loop (TTFL) comprising the Period1, 2 (Per1, 2), Cryptochrome1, 2 (Cry1, 2), and Brain and Muscle ARNT-Like 1 (Bmal1) genes. The robustness of the TTFL is attributed to genetic redundancy among some essential clock genes, deterring genetic studies on molecular clocks using genome editing targeting single genes. To manipulate multiple clock genes in a streamlined and efficient manner, we developed a CRISPR-Cas9-based single adeno-associated viral (AAV) system targeting the circadian clock (CSAC) for essential clock genes including Pers, Crys, or Bmal1. First, we tested several single guide RNAs (sgRNAs) targeting individual clock genes in silico and validated their efficiency in Neuro2a cells. To target multiple genes, multiplex sgRNA plasmids were constructed using Golden Gate assembly and packaged into AAVs. CSAC efficiency was evident through protein downregulation in vitro and ablated molecular oscillation ex vivo. We also measured the efficiency of CSAC in vivo by assessing circadian rhythms after injecting CSAC into the suprachiasmatic nuclei of Cas9-expressing knock-in mice. Circadian locomotor activity and body temperature rhythms were severely disrupted in these mice, indicating that our CSAC is a simple yet powerful tool for investigating the molecular clock in vivo. © 2021, The Author(s).
URI
http://hdl.handle.net/20.500.11750/13491
DOI
10.1038/s41598-021-82287-0
Publisher
Nature Publishing Group
Related Researcher
  • Author Choe, Han Kyoung Laboratory of Animal Behavior and Circadian rhythm
  • Research Interests Modulation of neural circuit; Circadian regulation of behavior and perception; Neurotechnology
Files:
Collection:
Department of Brain SciencesLaboratory of Animal Behavior and Circadian rhythm1. Journal Articles


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