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Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences

In CRISPR genome editing, CRISPR proteins form ribonucleoprotein complexes with guide RNAs to bind and cleave the target DNAs with complete sequence complementarity. CRISPR genome editing has a high potential for use in precision gene therapy for various diseases, including cancer and genetic disord...

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Autores principales: Bae, Taegeun, Kim, Hanseop, Kim, Jeong Hee, Kim, Yong Jun, Lee, Seung Hwan, Ham, Byung-Joo, Hur, Junho K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982904/
https://www.ncbi.nlm.nih.gov/pubmed/31877894
http://dx.doi.org/10.3390/molecules25010052
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author Bae, Taegeun
Kim, Hanseop
Kim, Jeong Hee
Kim, Yong Jun
Lee, Seung Hwan
Ham, Byung-Joo
Hur, Junho K.
author_facet Bae, Taegeun
Kim, Hanseop
Kim, Jeong Hee
Kim, Yong Jun
Lee, Seung Hwan
Ham, Byung-Joo
Hur, Junho K.
author_sort Bae, Taegeun
collection PubMed
description In CRISPR genome editing, CRISPR proteins form ribonucleoprotein complexes with guide RNAs to bind and cleave the target DNAs with complete sequence complementarity. CRISPR genome editing has a high potential for use in precision gene therapy for various diseases, including cancer and genetic disorders, which are caused by DNA mutations within the genome. However, several studies have shown that targeting the DNA via sequence complementarity is imperfect and subject to unintended genome editing of other genomic loci with similar sequences. These off-target problems pose critical safety issues in the therapeutic applications of CRISPR technology, with particular concerns in terms of the genome editing of pathogenic point mutations, where non-mutant alleles can become an off-target with only a one-base difference. In this study, we sought to assess a novel CRISPR genome editing technique that has been proposed to achieve a high specificity by positioning the mismatches within the protospacer adjacent motif (PAM) sequence. To this end, we compared the genome editing specificities of the PAM-based and conventional methods on an oncogenic single-base mutation in the endothelial growth factor receptor (EGFR). The results indicated that the PAM-based method provided a significantly increased genome editing specificity for pathogenic mutant alleles with single-base precision.
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spelling pubmed-69829042020-02-06 Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences Bae, Taegeun Kim, Hanseop Kim, Jeong Hee Kim, Yong Jun Lee, Seung Hwan Ham, Byung-Joo Hur, Junho K. Molecules Article In CRISPR genome editing, CRISPR proteins form ribonucleoprotein complexes with guide RNAs to bind and cleave the target DNAs with complete sequence complementarity. CRISPR genome editing has a high potential for use in precision gene therapy for various diseases, including cancer and genetic disorders, which are caused by DNA mutations within the genome. However, several studies have shown that targeting the DNA via sequence complementarity is imperfect and subject to unintended genome editing of other genomic loci with similar sequences. These off-target problems pose critical safety issues in the therapeutic applications of CRISPR technology, with particular concerns in terms of the genome editing of pathogenic point mutations, where non-mutant alleles can become an off-target with only a one-base difference. In this study, we sought to assess a novel CRISPR genome editing technique that has been proposed to achieve a high specificity by positioning the mismatches within the protospacer adjacent motif (PAM) sequence. To this end, we compared the genome editing specificities of the PAM-based and conventional methods on an oncogenic single-base mutation in the endothelial growth factor receptor (EGFR). The results indicated that the PAM-based method provided a significantly increased genome editing specificity for pathogenic mutant alleles with single-base precision. MDPI 2019-12-22 /pmc/articles/PMC6982904/ /pubmed/31877894 http://dx.doi.org/10.3390/molecules25010052 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Bae, Taegeun
Kim, Hanseop
Kim, Jeong Hee
Kim, Yong Jun
Lee, Seung Hwan
Ham, Byung-Joo
Hur, Junho K.
Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences
title Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences
title_full Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences
title_fullStr Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences
title_full_unstemmed Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences
title_short Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences
title_sort specificity assessment of crispr genome editing of oncogenic egfr point mutation with single-base differences
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982904/
https://www.ncbi.nlm.nih.gov/pubmed/31877894
http://dx.doi.org/10.3390/molecules25010052
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