Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9

Here, we present a multiplexed assay for variant effect protocol to assess the functional impact of all possible genetic variations within a particular genomic region. We describe steps for saturation genome editing by designing and cloning of single-guide RNA (sgRNA). We then detail steps for nucle...

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Autores principales: Sahu, Sounak, Sullivan, Teresa, Southon, Eileen, Caylor, Dylan, Geh, Josephine, Sharan, Shyam K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10658368/
https://www.ncbi.nlm.nih.gov/pubmed/37948185
http://dx.doi.org/10.1016/j.xpro.2023.102702
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author Sahu, Sounak
Sullivan, Teresa
Southon, Eileen
Caylor, Dylan
Geh, Josephine
Sharan, Shyam K.
author_facet Sahu, Sounak
Sullivan, Teresa
Southon, Eileen
Caylor, Dylan
Geh, Josephine
Sharan, Shyam K.
author_sort Sahu, Sounak
collection PubMed
description Here, we present a multiplexed assay for variant effect protocol to assess the functional impact of all possible genetic variations within a particular genomic region. We describe steps for saturation genome editing by designing and cloning of single-guide RNA (sgRNA). We then detail steps for nucleofection of sgRNA, testing drug response on variants, and amplification of genomic DNA for next-generation sequencing. For complete details on the use and execution of this protocol, please refer to Sahu et al.(1)
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spelling pubmed-106583682023-11-09 Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9 Sahu, Sounak Sullivan, Teresa Southon, Eileen Caylor, Dylan Geh, Josephine Sharan, Shyam K. STAR Protoc Protocol Here, we present a multiplexed assay for variant effect protocol to assess the functional impact of all possible genetic variations within a particular genomic region. We describe steps for saturation genome editing by designing and cloning of single-guide RNA (sgRNA). We then detail steps for nucleofection of sgRNA, testing drug response on variants, and amplification of genomic DNA for next-generation sequencing. For complete details on the use and execution of this protocol, please refer to Sahu et al.(1) Elsevier 2023-11-09 /pmc/articles/PMC10658368/ /pubmed/37948185 http://dx.doi.org/10.1016/j.xpro.2023.102702 Text en © 2023. https://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 Protocol
Sahu, Sounak
Sullivan, Teresa
Southon, Eileen
Caylor, Dylan
Geh, Josephine
Sharan, Shyam K.
Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9
title Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9
title_full Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9
title_fullStr Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9
title_full_unstemmed Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9
title_short Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9
title_sort protocol for the saturation and multiplexing of genetic variants using crispr-cas9
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10658368/
https://www.ncbi.nlm.nih.gov/pubmed/37948185
http://dx.doi.org/10.1016/j.xpro.2023.102702
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