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  • 标题:Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes
  • 本地全文:下载
  • 作者:Boil Kim ; Jihoon Kim ; Minjeong Chun
  • 期刊名称:Scientific Reports
  • 电子版ISSN:2045-2322
  • 出版年度:2021
  • 卷号:11
  • 期号:1
  • 页码:2575
  • DOI:10.1038/s41598-021-82287-0
  • 出版社:Springer Nature
  • 摘要: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 C RISPR-Cas9-based s ingle a deno-associated viral (AAV) system targeting the c ircadian clock (CSAC) for essential clock genes including Per s, Cry s, 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.
  • 其他摘要: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 C RISPR-Cas9-based s ingle a deno-associated viral (AAV) system targeting the c ircadian clock (CSAC) for essential clock genes including Per s, Cry s, 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.
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