LATE–a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects

Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although...
Ausführliche Beschreibung

Gespeichert in:

Autor*in:	Dawid Głów [verfasserIn] Simon Meyer [verfasserIn] Irene García Roldán [verfasserIn] Lara Marie Akingunsade [verfasserIn] Kristoffer Riecken [verfasserIn] Boris Fehse [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2021

Schlagwörter:	CRISPR/Cas genome editing off-targets side effects gene therapy cellular assay

Übergeordnetes Werk:	In: Molecular Therapy: Methods & Clinical Development - Elsevier, 2015, 22(2021), Seite 249-262
Übergeordnetes Werk:	volume:22 ; year:2021 ; pages:249-262

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.1016/j.omtm.2021.07.004

Katalog-ID:	DOAJ019947658

Internformat


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allfields	10.1016/j.omtm.2021.07.004 doi (DE-627)DOAJ019947658 (DE-599)DOAJ6f33396e84f94dfc9ac886fa0a5d9a8b DE-627 ger DE-627 rakwb eng QH426-470 QH573-671 Dawid Głów verfasserin aut LATE–a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (<0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies. CRISPR/Cas genome editing off-targets side effects gene therapy cellular assay Genetics Cytology Simon Meyer verfasserin aut Irene García Roldán verfasserin aut Lara Marie Akingunsade verfasserin aut Kristoffer Riecken verfasserin aut Boris Fehse verfasserin aut In Molecular Therapy: Methods & Clinical Development Elsevier, 2015 22(2021), Seite 249-262 (DE-627)863823203 (DE-600)2863173-0 23290501 nnns volume:22 year:2021 pages:249-262 https://doi.org/10.1016/j.omtm.2021.07.004 kostenfrei https://doaj.org/article/6f33396e84f94dfc9ac886fa0a5d9a8b kostenfrei http://www.sciencedirect.com/science/article/pii/S2329050121001212 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 22 2021 249-262
spelling	10.1016/j.omtm.2021.07.004 doi (DE-627)DOAJ019947658 (DE-599)DOAJ6f33396e84f94dfc9ac886fa0a5d9a8b DE-627 ger DE-627 rakwb eng QH426-470 QH573-671 Dawid Głów verfasserin aut LATE–a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (<0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies. CRISPR/Cas genome editing off-targets side effects gene therapy cellular assay Genetics Cytology Simon Meyer verfasserin aut Irene García Roldán verfasserin aut Lara Marie Akingunsade verfasserin aut Kristoffer Riecken verfasserin aut Boris Fehse verfasserin aut In Molecular Therapy: Methods & Clinical Development Elsevier, 2015 22(2021), Seite 249-262 (DE-627)863823203 (DE-600)2863173-0 23290501 nnns volume:22 year:2021 pages:249-262 https://doi.org/10.1016/j.omtm.2021.07.004 kostenfrei https://doaj.org/article/6f33396e84f94dfc9ac886fa0a5d9a8b kostenfrei http://www.sciencedirect.com/science/article/pii/S2329050121001212 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 22 2021 249-262
allfields_unstemmed	10.1016/j.omtm.2021.07.004 doi (DE-627)DOAJ019947658 (DE-599)DOAJ6f33396e84f94dfc9ac886fa0a5d9a8b DE-627 ger DE-627 rakwb eng QH426-470 QH573-671 Dawid Głów verfasserin aut LATE–a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (<0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies. CRISPR/Cas genome editing off-targets side effects gene therapy cellular assay Genetics Cytology Simon Meyer verfasserin aut Irene García Roldán verfasserin aut Lara Marie Akingunsade verfasserin aut Kristoffer Riecken verfasserin aut Boris Fehse verfasserin aut In Molecular Therapy: Methods & Clinical Development Elsevier, 2015 22(2021), Seite 249-262 (DE-627)863823203 (DE-600)2863173-0 23290501 nnns volume:22 year:2021 pages:249-262 https://doi.org/10.1016/j.omtm.2021.07.004 kostenfrei https://doaj.org/article/6f33396e84f94dfc9ac886fa0a5d9a8b kostenfrei http://www.sciencedirect.com/science/article/pii/S2329050121001212 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 22 2021 249-262
allfieldsGer	10.1016/j.omtm.2021.07.004 doi (DE-627)DOAJ019947658 (DE-599)DOAJ6f33396e84f94dfc9ac886fa0a5d9a8b DE-627 ger DE-627 rakwb eng QH426-470 QH573-671 Dawid Głów verfasserin aut LATE–a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (<0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies. CRISPR/Cas genome editing off-targets side effects gene therapy cellular assay Genetics Cytology Simon Meyer verfasserin aut Irene García Roldán verfasserin aut Lara Marie Akingunsade verfasserin aut Kristoffer Riecken verfasserin aut Boris Fehse verfasserin aut In Molecular Therapy: Methods & Clinical Development Elsevier, 2015 22(2021), Seite 249-262 (DE-627)863823203 (DE-600)2863173-0 23290501 nnns volume:22 year:2021 pages:249-262 https://doi.org/10.1016/j.omtm.2021.07.004 kostenfrei https://doaj.org/article/6f33396e84f94dfc9ac886fa0a5d9a8b kostenfrei http://www.sciencedirect.com/science/article/pii/S2329050121001212 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 22 2021 249-262
allfieldsSound	10.1016/j.omtm.2021.07.004 doi (DE-627)DOAJ019947658 (DE-599)DOAJ6f33396e84f94dfc9ac886fa0a5d9a8b DE-627 ger DE-627 rakwb eng QH426-470 QH573-671 Dawid Głów verfasserin aut LATE–a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (<0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies. CRISPR/Cas genome editing off-targets side effects gene therapy cellular assay Genetics Cytology Simon Meyer verfasserin aut Irene García Roldán verfasserin aut Lara Marie Akingunsade verfasserin aut Kristoffer Riecken verfasserin aut Boris Fehse verfasserin aut In Molecular Therapy: Methods & Clinical Development Elsevier, 2015 22(2021), Seite 249-262 (DE-627)863823203 (DE-600)2863173-0 23290501 nnns volume:22 year:2021 pages:249-262 https://doi.org/10.1016/j.omtm.2021.07.004 kostenfrei https://doaj.org/article/6f33396e84f94dfc9ac886fa0a5d9a8b kostenfrei http://www.sciencedirect.com/science/article/pii/S2329050121001212 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 22 2021 249-262
language	English
source	In Molecular Therapy: Methods & Clinical Development 22(2021), Seite 249-262 volume:22 year:2021 pages:249-262
sourceStr	In Molecular Therapy: Methods & Clinical Development 22(2021), Seite 249-262 volume:22 year:2021 pages:249-262
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	CRISPR/Cas genome editing off-targets side effects gene therapy cellular assay Genetics Cytology
isfreeaccess_bool	true
container_title	Molecular Therapy: Methods & Clinical Development
authorswithroles_txt_mv	Dawid Głów @@aut@@ Simon Meyer @@aut@@ Irene García Roldán @@aut@@ Lara Marie Akingunsade @@aut@@ Kristoffer Riecken @@aut@@ Boris Fehse @@aut@@
publishDateDaySort_date	2021-01-01T00:00:00Z
hierarchy_top_id	863823203
id	DOAJ019947658
language_de	englisch
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callnumber-first	Q - Science
author	Dawid Głów
spellingShingle	Dawid Głów misc QH426-470 misc QH573-671 misc CRISPR/Cas misc genome editing misc off-targets misc side effects misc gene therapy misc cellular assay misc Genetics misc Cytology LATE–a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects
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topic_title	QH426-470 QH573-671 LATE–a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects CRISPR/Cas genome editing off-targets side effects gene therapy cellular assay
topic	misc QH426-470 misc QH573-671 misc CRISPR/Cas misc genome editing misc off-targets misc side effects misc gene therapy misc cellular assay misc Genetics misc Cytology
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title	LATE–a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects
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title_full	LATE–a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects
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author_browse	Dawid Głów Simon Meyer Irene García Roldán Lara Marie Akingunsade Kristoffer Riecken Boris Fehse
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title_sort	late–a novel sensitive cell-based assay for the study of crispr/cas9-related long-term adverse treatment effects
callnumber	QH426-470
title_auth	LATE–a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects
abstract	Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (<0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies.
abstractGer	Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (<0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies.
abstract_unstemmed	Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (<0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies.
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title_short	LATE–a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects
url	https://doi.org/10.1016/j.omtm.2021.07.004 https://doaj.org/article/6f33396e84f94dfc9ac886fa0a5d9a8b http://www.sciencedirect.com/science/article/pii/S2329050121001212 https://doaj.org/toc/2329-0501
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LATE–a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects

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