A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing

Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expres...
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Autor*in:	Ang Li [verfasserIn] Ciaran M. Lee [verfasserIn] Ayrea E. Hurley [verfasserIn] Kelsey E. Jarrett [verfasserIn] Marco De Giorgi [verfasserIn] Weiqi Lu [verfasserIn] Karol S. Balderrama [verfasserIn] Alexandria M. Doerfler [verfasserIn] Harshavardhan Deshmukh [verfasserIn] Anirban Ray [verfasserIn] Gang Bao [verfasserIn] William R. Lagor [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2019

Übergeordnetes Werk:	In: Molecular Therapy: Methods & Clinical Development - Elsevier, 2015, 12(2019), Seite 111-122
Übergeordnetes Werk:	volume:12 ; year:2019 ; pages:111-122

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.1016/j.omtm.2018.11.009

Katalog-ID:	DOAJ040124258

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520			\|a Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans. Keywords: CRISPR/Cas9, adeno-associated virus, AAV, in vivo delivery, self-deleting, somatic genome editing, liver, AAV-CRISPR, gene therapy
653		0	\|a Genetics
653		0	\|a Cytology
700	0		\|a Ciaran M. Lee \|e verfasserin \|4 aut
700	0		\|a Ayrea E. Hurley \|e verfasserin \|4 aut
700	0		\|a Kelsey E. Jarrett \|e verfasserin \|4 aut
700	0		\|a Marco De Giorgi \|e verfasserin \|4 aut
700	0		\|a Weiqi Lu \|e verfasserin \|4 aut
700	0		\|a Karol S. Balderrama \|e verfasserin \|4 aut
700	0		\|a Alexandria M. Doerfler \|e verfasserin \|4 aut
700	0		\|a Harshavardhan Deshmukh \|e verfasserin \|4 aut
700	0		\|a Anirban Ray \|e verfasserin \|4 aut
700	0		\|a Gang Bao \|e verfasserin \|4 aut
700	0		\|a William R. Lagor \|e verfasserin \|4 aut
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publishDate	2019
allfields	10.1016/j.omtm.2018.11.009 doi (DE-627)DOAJ040124258 (DE-599)DOAJ312deba985e94bf79a24b835737e75c8 DE-627 ger DE-627 rakwb eng QH426-470 QH573-671 Ang Li verfasserin aut A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans. Keywords: CRISPR/Cas9, adeno-associated virus, AAV, in vivo delivery, self-deleting, somatic genome editing, liver, AAV-CRISPR, gene therapy Genetics Cytology Ciaran M. Lee verfasserin aut Ayrea E. Hurley verfasserin aut Kelsey E. Jarrett verfasserin aut Marco De Giorgi verfasserin aut Weiqi Lu verfasserin aut Karol S. Balderrama verfasserin aut Alexandria M. Doerfler verfasserin aut Harshavardhan Deshmukh verfasserin aut Anirban Ray verfasserin aut Gang Bao verfasserin aut William R. Lagor verfasserin aut In Molecular Therapy: Methods & Clinical Development Elsevier, 2015 12(2019), Seite 111-122 (DE-627)863823203 (DE-600)2863173-0 23290501 nnns volume:12 year:2019 pages:111-122 https://doi.org/10.1016/j.omtm.2018.11.009 kostenfrei https://doaj.org/article/312deba985e94bf79a24b835737e75c8 kostenfrei http://www.sciencedirect.com/science/article/pii/S2329050118301219 kostenfrei https://doaj.org/toc/2329-0501 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2110 GBV_ILN_2112 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2153 GBV_ILN_2190 GBV_ILN_2336 GBV_ILN_2470 GBV_ILN_2507 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4393 GBV_ILN_4700 AR 12 2019 111-122
spelling	10.1016/j.omtm.2018.11.009 doi (DE-627)DOAJ040124258 (DE-599)DOAJ312deba985e94bf79a24b835737e75c8 DE-627 ger DE-627 rakwb eng QH426-470 QH573-671 Ang Li verfasserin aut A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans. Keywords: CRISPR/Cas9, adeno-associated virus, AAV, in vivo delivery, self-deleting, somatic genome editing, liver, AAV-CRISPR, gene therapy Genetics Cytology Ciaran M. Lee verfasserin aut Ayrea E. Hurley verfasserin aut Kelsey E. Jarrett verfasserin aut Marco De Giorgi verfasserin aut Weiqi Lu verfasserin aut Karol S. Balderrama verfasserin aut Alexandria M. Doerfler verfasserin aut Harshavardhan Deshmukh verfasserin aut Anirban Ray verfasserin aut Gang Bao verfasserin aut William R. Lagor verfasserin aut In Molecular Therapy: Methods & Clinical Development Elsevier, 2015 12(2019), Seite 111-122 (DE-627)863823203 (DE-600)2863173-0 23290501 nnns volume:12 year:2019 pages:111-122 https://doi.org/10.1016/j.omtm.2018.11.009 kostenfrei https://doaj.org/article/312deba985e94bf79a24b835737e75c8 kostenfrei http://www.sciencedirect.com/science/article/pii/S2329050118301219 kostenfrei https://doaj.org/toc/2329-0501 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2110 GBV_ILN_2112 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2153 GBV_ILN_2190 GBV_ILN_2336 GBV_ILN_2470 GBV_ILN_2507 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4393 GBV_ILN_4700 AR 12 2019 111-122
allfields_unstemmed	10.1016/j.omtm.2018.11.009 doi (DE-627)DOAJ040124258 (DE-599)DOAJ312deba985e94bf79a24b835737e75c8 DE-627 ger DE-627 rakwb eng QH426-470 QH573-671 Ang Li verfasserin aut A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans. Keywords: CRISPR/Cas9, adeno-associated virus, AAV, in vivo delivery, self-deleting, somatic genome editing, liver, AAV-CRISPR, gene therapy Genetics Cytology Ciaran M. Lee verfasserin aut Ayrea E. Hurley verfasserin aut Kelsey E. Jarrett verfasserin aut Marco De Giorgi verfasserin aut Weiqi Lu verfasserin aut Karol S. Balderrama verfasserin aut Alexandria M. Doerfler verfasserin aut Harshavardhan Deshmukh verfasserin aut Anirban Ray verfasserin aut Gang Bao verfasserin aut William R. Lagor verfasserin aut In Molecular Therapy: Methods & Clinical Development Elsevier, 2015 12(2019), Seite 111-122 (DE-627)863823203 (DE-600)2863173-0 23290501 nnns volume:12 year:2019 pages:111-122 https://doi.org/10.1016/j.omtm.2018.11.009 kostenfrei https://doaj.org/article/312deba985e94bf79a24b835737e75c8 kostenfrei http://www.sciencedirect.com/science/article/pii/S2329050118301219 kostenfrei https://doaj.org/toc/2329-0501 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2110 GBV_ILN_2112 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2153 GBV_ILN_2190 GBV_ILN_2336 GBV_ILN_2470 GBV_ILN_2507 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4393 GBV_ILN_4700 AR 12 2019 111-122
allfieldsGer	10.1016/j.omtm.2018.11.009 doi (DE-627)DOAJ040124258 (DE-599)DOAJ312deba985e94bf79a24b835737e75c8 DE-627 ger DE-627 rakwb eng QH426-470 QH573-671 Ang Li verfasserin aut A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans. Keywords: CRISPR/Cas9, adeno-associated virus, AAV, in vivo delivery, self-deleting, somatic genome editing, liver, AAV-CRISPR, gene therapy Genetics Cytology Ciaran M. Lee verfasserin aut Ayrea E. Hurley verfasserin aut Kelsey E. Jarrett verfasserin aut Marco De Giorgi verfasserin aut Weiqi Lu verfasserin aut Karol S. Balderrama verfasserin aut Alexandria M. Doerfler verfasserin aut Harshavardhan Deshmukh verfasserin aut Anirban Ray verfasserin aut Gang Bao verfasserin aut William R. Lagor verfasserin aut In Molecular Therapy: Methods & Clinical Development Elsevier, 2015 12(2019), Seite 111-122 (DE-627)863823203 (DE-600)2863173-0 23290501 nnns volume:12 year:2019 pages:111-122 https://doi.org/10.1016/j.omtm.2018.11.009 kostenfrei https://doaj.org/article/312deba985e94bf79a24b835737e75c8 kostenfrei http://www.sciencedirect.com/science/article/pii/S2329050118301219 kostenfrei https://doaj.org/toc/2329-0501 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2110 GBV_ILN_2112 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2153 GBV_ILN_2190 GBV_ILN_2336 GBV_ILN_2470 GBV_ILN_2507 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4393 GBV_ILN_4700 AR 12 2019 111-122
allfieldsSound	10.1016/j.omtm.2018.11.009 doi (DE-627)DOAJ040124258 (DE-599)DOAJ312deba985e94bf79a24b835737e75c8 DE-627 ger DE-627 rakwb eng QH426-470 QH573-671 Ang Li verfasserin aut A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans. Keywords: CRISPR/Cas9, adeno-associated virus, AAV, in vivo delivery, self-deleting, somatic genome editing, liver, AAV-CRISPR, gene therapy Genetics Cytology Ciaran M. Lee verfasserin aut Ayrea E. Hurley verfasserin aut Kelsey E. Jarrett verfasserin aut Marco De Giorgi verfasserin aut Weiqi Lu verfasserin aut Karol S. Balderrama verfasserin aut Alexandria M. Doerfler verfasserin aut Harshavardhan Deshmukh verfasserin aut Anirban Ray verfasserin aut Gang Bao verfasserin aut William R. Lagor verfasserin aut In Molecular Therapy: Methods & Clinical Development Elsevier, 2015 12(2019), Seite 111-122 (DE-627)863823203 (DE-600)2863173-0 23290501 nnns volume:12 year:2019 pages:111-122 https://doi.org/10.1016/j.omtm.2018.11.009 kostenfrei https://doaj.org/article/312deba985e94bf79a24b835737e75c8 kostenfrei http://www.sciencedirect.com/science/article/pii/S2329050118301219 kostenfrei https://doaj.org/toc/2329-0501 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2110 GBV_ILN_2112 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2153 GBV_ILN_2190 GBV_ILN_2336 GBV_ILN_2470 GBV_ILN_2507 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4393 GBV_ILN_4700 AR 12 2019 111-122
language	English
source	In Molecular Therapy: Methods & Clinical Development 12(2019), Seite 111-122 volume:12 year:2019 pages:111-122
sourceStr	In Molecular Therapy: Methods & Clinical Development 12(2019), Seite 111-122 volume:12 year:2019 pages:111-122
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	Genetics Cytology
isfreeaccess_bool	true
container_title	Molecular Therapy: Methods & Clinical Development
authorswithroles_txt_mv	Ang Li @@aut@@ Ciaran M. Lee @@aut@@ Ayrea E. Hurley @@aut@@ Kelsey E. Jarrett @@aut@@ Marco De Giorgi @@aut@@ Weiqi Lu @@aut@@ Karol S. Balderrama @@aut@@ Alexandria M. Doerfler @@aut@@ Harshavardhan Deshmukh @@aut@@ Anirban Ray @@aut@@ Gang Bao @@aut@@ William R. Lagor @@aut@@
publishDateDaySort_date	2019-01-01T00:00:00Z
hierarchy_top_id	863823203
id	DOAJ040124258
language_de	englisch
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callnumber-first	Q - Science
author	Ang Li
spellingShingle	Ang Li misc QH426-470 misc QH573-671 misc Genetics misc Cytology A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing
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topic_title	QH426-470 QH573-671 A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing
topic	misc QH426-470 misc QH573-671 misc Genetics misc Cytology
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title	A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing
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title_full	A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing
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author_browse	Ang Li Ciaran M. Lee Ayrea E. Hurley Kelsey E. Jarrett Marco De Giorgi Weiqi Lu Karol S. Balderrama Alexandria M. Doerfler Harshavardhan Deshmukh Anirban Ray Gang Bao William R. Lagor
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title_sort	self-deleting aav-crispr system for in vivo genome editing
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title_auth	A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing
abstract	Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans. Keywords: CRISPR/Cas9, adeno-associated virus, AAV, in vivo delivery, self-deleting, somatic genome editing, liver, AAV-CRISPR, gene therapy
abstractGer	Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans. Keywords: CRISPR/Cas9, adeno-associated virus, AAV, in vivo delivery, self-deleting, somatic genome editing, liver, AAV-CRISPR, gene therapy
abstract_unstemmed	Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans. Keywords: CRISPR/Cas9, adeno-associated virus, AAV, in vivo delivery, self-deleting, somatic genome editing, liver, AAV-CRISPR, gene therapy
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title_short	A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing
url	https://doi.org/10.1016/j.omtm.2018.11.009 https://doaj.org/article/312deba985e94bf79a24b835737e75c8 http://www.sciencedirect.com/science/article/pii/S2329050118301219 https://doaj.org/toc/2329-0501
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up_date	2024-07-04T02:01:35.599Z
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A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing

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