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

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 clo...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Boil Kim, Jihoon Kim, Minjeong Chun, Inah Park, Damhyeon Kwak, Mijung Choi, Kyungjin Kim, Han Kyoung Choe
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/1542793254f346c39d8097beb11fb52d
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:1542793254f346c39d8097beb11fb52d
record_format	dspace
spelling	oai:doaj.org-article:1542793254f346c39d8097beb11fb52d2021-12-02T13:57:25ZMultiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes10.1038/s41598-021-82287-02045-2322https://doaj.org/article/1542793254f346c39d8097beb11fb52d2021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-82287-0https://doaj.org/toc/2045-2322Abstract 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.Boil KimJihoon KimMinjeong ChunInah ParkDamhyeon KwakMijung ChoiKyungjin KimHan Kyoung ChoeNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Boil Kim Jihoon Kim Minjeong Chun Inah Park Damhyeon Kwak Mijung Choi Kyungjin Kim Han Kyoung Choe Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes
description	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.
format	article
author	Boil Kim Jihoon Kim Minjeong Chun Inah Park Damhyeon Kwak Mijung Choi Kyungjin Kim Han Kyoung Choe
author_facet	Boil Kim Jihoon Kim Minjeong Chun Inah Park Damhyeon Kwak Mijung Choi Kyungjin Kim Han Kyoung Choe
author_sort	Boil Kim
title	Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes
title_short	Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes
title_full	Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes
title_fullStr	Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes
title_full_unstemmed	Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes
title_sort	multiplexed crispr-cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/1542793254f346c39d8097beb11fb52d
work_keys_str_mv	AT boilkim multiplexedcrisprcas9systeminasingleadenoassociatedvirustosimultaneouslyknockoutredundantclockgenes AT jihoonkim multiplexedcrisprcas9systeminasingleadenoassociatedvirustosimultaneouslyknockoutredundantclockgenes AT minjeongchun multiplexedcrisprcas9systeminasingleadenoassociatedvirustosimultaneouslyknockoutredundantclockgenes AT inahpark multiplexedcrisprcas9systeminasingleadenoassociatedvirustosimultaneouslyknockoutredundantclockgenes AT damhyeonkwak multiplexedcrisprcas9systeminasingleadenoassociatedvirustosimultaneouslyknockoutredundantclockgenes AT mijungchoi multiplexedcrisprcas9systeminasingleadenoassociatedvirustosimultaneouslyknockoutredundantclockgenes AT kyungjinkim multiplexedcrisprcas9systeminasingleadenoassociatedvirustosimultaneouslyknockoutredundantclockgenes AT hankyoungchoe multiplexedcrisprcas9systeminasingleadenoassociatedvirustosimultaneouslyknockoutredundantclockgenes
_version_	1718392304950050816

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

Ejemplares similares