Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair

Abstract CRISPR–Cas proteins are RNA-guided nucleases used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing an exogenous DNA template during r...

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Autores principales:	Mollie S. Schubert, Bernice Thommandru, Jessica Woodley, Rolf Turk, Shuqi Yan, Gavin Kurgan, Matthew S. McNeill, Garrett R. Rettig
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/beb41c1f9a884d0690dce2597cdadd0e
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spelling	oai:doaj.org-article:beb41c1f9a884d0690dce2597cdadd0e2021-12-02T17:17:38ZOptimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair10.1038/s41598-021-98965-y2045-2322https://doaj.org/article/beb41c1f9a884d0690dce2597cdadd0e2021-09-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-98965-yhttps://doaj.org/toc/2045-2322Abstract CRISPR–Cas proteins are RNA-guided nucleases used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing an exogenous DNA template during repair allows for the intentional, precise incorporation of a desired mutation via the HDR pathway. However, rates of repair by HDR are often slow compared to the more rapid but less accurate NHEJ-mediated repair. Here, we describe comprehensive design considerations and optimized methods for highly efficient HDR using single-stranded oligodeoxynucleotide (ssODN) donor templates for several CRISPR–Cas systems including S.p. Cas9, S.p. Cas9 D10A nickase, and A.s. Cas12a delivered as ribonucleoprotein (RNP) complexes. Features relating to guide RNA selection, donor strand preference, and incorporation of blocking mutations in the donor template to prevent re-cleavage were investigated and were implemented in a novel online tool for HDR donor template design. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Tool availability: https://www.idtdna.com/HDRMollie S. SchubertBernice ThommandruJessica WoodleyRolf TurkShuqi YanGavin KurganMatthew S. McNeillGarrett R. RettigNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-15 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Mollie S. Schubert Bernice Thommandru Jessica Woodley Rolf Turk Shuqi Yan Gavin Kurgan Matthew S. McNeill Garrett R. Rettig Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair
description	Abstract CRISPR–Cas proteins are RNA-guided nucleases used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing an exogenous DNA template during repair allows for the intentional, precise incorporation of a desired mutation via the HDR pathway. However, rates of repair by HDR are often slow compared to the more rapid but less accurate NHEJ-mediated repair. Here, we describe comprehensive design considerations and optimized methods for highly efficient HDR using single-stranded oligodeoxynucleotide (ssODN) donor templates for several CRISPR–Cas systems including S.p. Cas9, S.p. Cas9 D10A nickase, and A.s. Cas12a delivered as ribonucleoprotein (RNP) complexes. Features relating to guide RNA selection, donor strand preference, and incorporation of blocking mutations in the donor template to prevent re-cleavage were investigated and were implemented in a novel online tool for HDR donor template design. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Tool availability: https://www.idtdna.com/HDR
format	article
author	Mollie S. Schubert Bernice Thommandru Jessica Woodley Rolf Turk Shuqi Yan Gavin Kurgan Matthew S. McNeill Garrett R. Rettig
author_facet	Mollie S. Schubert Bernice Thommandru Jessica Woodley Rolf Turk Shuqi Yan Gavin Kurgan Matthew S. McNeill Garrett R. Rettig
author_sort	Mollie S. Schubert
title	Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair
title_short	Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair
title_full	Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair
title_fullStr	Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair
title_full_unstemmed	Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair
title_sort	optimized design parameters for crispr cas9 and cas12a homology-directed repair
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/beb41c1f9a884d0690dce2597cdadd0e
work_keys_str_mv	AT molliesschubert optimizeddesignparametersforcrisprcas9andcas12ahomologydirectedrepair AT bernicethommandru optimizeddesignparametersforcrisprcas9andcas12ahomologydirectedrepair AT jessicawoodley optimizeddesignparametersforcrisprcas9andcas12ahomologydirectedrepair AT rolfturk optimizeddesignparametersforcrisprcas9andcas12ahomologydirectedrepair AT shuqiyan optimizeddesignparametersforcrisprcas9andcas12ahomologydirectedrepair AT gavinkurgan optimizeddesignparametersforcrisprcas9andcas12ahomologydirectedrepair AT matthewsmcneill optimizeddesignparametersforcrisprcas9andcas12ahomologydirectedrepair AT garrettrrettig optimizeddesignparametersforcrisprcas9andcas12ahomologydirectedrepair
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Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair

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