Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

Abstract The powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it oc...

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Autores principales:	Yuu Asano, Kensuke Yamashita, Aoi Hasegawa, Takanori Ogasawara, Hoshie Iriki, Tetsuya Muramoto
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/4def373470b34e73acbf59ccbfd28663
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spelling	oai:doaj.org-article:4def373470b34e73acbf59ccbfd286632021-12-02T15:00:25ZKnock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum10.1038/s41598-021-89546-02045-2322https://doaj.org/article/4def373470b34e73acbf59ccbfd286632021-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-89546-0https://doaj.org/toc/2045-2322Abstract The powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.Yuu AsanoKensuke YamashitaAoi HasegawaTakanori OgasawaraHoshie IrikiTetsuya MuramotoNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yuu Asano Kensuke Yamashita Aoi Hasegawa Takanori Ogasawara Hoshie Iriki Tetsuya Muramoto Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum
description	Abstract The powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.
format	article
author	Yuu Asano Kensuke Yamashita Aoi Hasegawa Takanori Ogasawara Hoshie Iriki Tetsuya Muramoto
author_facet	Yuu Asano Kensuke Yamashita Aoi Hasegawa Takanori Ogasawara Hoshie Iriki Tetsuya Muramoto
author_sort	Yuu Asano
title	Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum
title_short	Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum
title_full	Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum
title_fullStr	Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum
title_full_unstemmed	Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum
title_sort	knock-in and precise nucleotide substitution using near-pamless engineered cas9 variants in dictyostelium discoideum
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/4def373470b34e73acbf59ccbfd28663
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Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

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