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Cloning-free CRISPR

We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each targ...

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Autores principales: Arbab, Mandana, Srinivasan, Sharanya, Hashimoto, Tatsunori, Geijsen, Niels, Sherwood, Richard I.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4649464/
https://www.ncbi.nlm.nih.gov/pubmed/26527385
http://dx.doi.org/10.1016/j.stemcr.2015.09.022
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author Arbab, Mandana
Srinivasan, Sharanya
Hashimoto, Tatsunori
Geijsen, Niels
Sherwood, Richard I.
author_facet Arbab, Mandana
Srinivasan, Sharanya
Hashimoto, Tatsunori
Geijsen, Niels
Sherwood, Richard I.
author_sort Arbab, Mandana
collection PubMed
description We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis.
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spelling pubmed-46494642015-12-11 Cloning-free CRISPR Arbab, Mandana Srinivasan, Sharanya Hashimoto, Tatsunori Geijsen, Niels Sherwood, Richard I. Stem Cell Reports Resource We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis. Elsevier 2015-10-29 /pmc/articles/PMC4649464/ /pubmed/26527385 http://dx.doi.org/10.1016/j.stemcr.2015.09.022 Text en © 2015 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Resource
Arbab, Mandana
Srinivasan, Sharanya
Hashimoto, Tatsunori
Geijsen, Niels
Sherwood, Richard I.
Cloning-free CRISPR
title Cloning-free CRISPR
title_full Cloning-free CRISPR
title_fullStr Cloning-free CRISPR
title_full_unstemmed Cloning-free CRISPR
title_short Cloning-free CRISPR
title_sort cloning-free crispr
topic Resource
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4649464/
https://www.ncbi.nlm.nih.gov/pubmed/26527385
http://dx.doi.org/10.1016/j.stemcr.2015.09.022
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