The position of the target site for engineered nucleases improves the aberrant mRNA clearance in in vivo genome editing

Engineered nucleases are widely used for creating frameshift or nonsense mutations in the target genes to eliminate gene functions. The resulting mRNAs carrying premature termination codons can be eliminated by nonsense-mediated mRNA decay. However, it is unclear how effective this process would be...

Descripción completa

Detalles Bibliográficos
Autores principales: Lee, Jae Hoon, Yu, Sungsook, Nam, Tae Wook, Roh, Jae-il, Jin, Young, Han, Jeong Pil, Cha, Ji-Young, Kim, Yoon Ki, Yeom, Su-Cheong, Nam, Ki Taek, Lee, Han-Woong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060192/
https://www.ncbi.nlm.nih.gov/pubmed/32144373
http://dx.doi.org/10.1038/s41598-020-61154-4
_version_ 1783504179616022528
author Lee, Jae Hoon
Yu, Sungsook
Nam, Tae Wook
Roh, Jae-il
Jin, Young
Han, Jeong Pil
Cha, Ji-Young
Kim, Yoon Ki
Yeom, Su-Cheong
Nam, Ki Taek
Lee, Han-Woong
author_facet Lee, Jae Hoon
Yu, Sungsook
Nam, Tae Wook
Roh, Jae-il
Jin, Young
Han, Jeong Pil
Cha, Ji-Young
Kim, Yoon Ki
Yeom, Su-Cheong
Nam, Ki Taek
Lee, Han-Woong
author_sort Lee, Jae Hoon
collection PubMed
description Engineered nucleases are widely used for creating frameshift or nonsense mutations in the target genes to eliminate gene functions. The resulting mRNAs carrying premature termination codons can be eliminated by nonsense-mediated mRNA decay. However, it is unclear how effective this process would be in vivo. Here, we found that the nonsense-mediated decay was unable to remove the mutant mRNAs in twelve out of sixteen homozygous mutant mice with frameshift mutations generated using engineered nucleases, which is far beyond what we expected. The frameshift mutant proteins translated by a single nucleotide deletion within the coding region were also detected in the p53 mutant mice. Furthermore, we showed that targeting the exons present downstream of the exons with a start codon or distant from ATG is relatively effective for eliminating mutant mRNAs in vivo, whereas the exons with a start codon are targeted to express the mutant mRNAs. Of the sixteen mutant mice generated, only four mutant mice targeting the downstream exons exhibited over 80% clearance of mutant mRNAs. Since the abnormal products, either mutant RNAs or mutant proteins, expressed by the target alleles might obscure the outcome of genome editing, these findings will provide insights in the improved performance of engineered nucleases when they are applied in vivo.
format Online
Article
Text
id pubmed-7060192
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher Nature Publishing Group UK
record_format MEDLINE/PubMed
spelling pubmed-70601922020-03-18 The position of the target site for engineered nucleases improves the aberrant mRNA clearance in in vivo genome editing Lee, Jae Hoon Yu, Sungsook Nam, Tae Wook Roh, Jae-il Jin, Young Han, Jeong Pil Cha, Ji-Young Kim, Yoon Ki Yeom, Su-Cheong Nam, Ki Taek Lee, Han-Woong Sci Rep Article Engineered nucleases are widely used for creating frameshift or nonsense mutations in the target genes to eliminate gene functions. The resulting mRNAs carrying premature termination codons can be eliminated by nonsense-mediated mRNA decay. However, it is unclear how effective this process would be in vivo. Here, we found that the nonsense-mediated decay was unable to remove the mutant mRNAs in twelve out of sixteen homozygous mutant mice with frameshift mutations generated using engineered nucleases, which is far beyond what we expected. The frameshift mutant proteins translated by a single nucleotide deletion within the coding region were also detected in the p53 mutant mice. Furthermore, we showed that targeting the exons present downstream of the exons with a start codon or distant from ATG is relatively effective for eliminating mutant mRNAs in vivo, whereas the exons with a start codon are targeted to express the mutant mRNAs. Of the sixteen mutant mice generated, only four mutant mice targeting the downstream exons exhibited over 80% clearance of mutant mRNAs. Since the abnormal products, either mutant RNAs or mutant proteins, expressed by the target alleles might obscure the outcome of genome editing, these findings will provide insights in the improved performance of engineered nucleases when they are applied in vivo. Nature Publishing Group UK 2020-03-06 /pmc/articles/PMC7060192/ /pubmed/32144373 http://dx.doi.org/10.1038/s41598-020-61154-4 Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Lee, Jae Hoon
Yu, Sungsook
Nam, Tae Wook
Roh, Jae-il
Jin, Young
Han, Jeong Pil
Cha, Ji-Young
Kim, Yoon Ki
Yeom, Su-Cheong
Nam, Ki Taek
Lee, Han-Woong
The position of the target site for engineered nucleases improves the aberrant mRNA clearance in in vivo genome editing
title The position of the target site for engineered nucleases improves the aberrant mRNA clearance in in vivo genome editing
title_full The position of the target site for engineered nucleases improves the aberrant mRNA clearance in in vivo genome editing
title_fullStr The position of the target site for engineered nucleases improves the aberrant mRNA clearance in in vivo genome editing
title_full_unstemmed The position of the target site for engineered nucleases improves the aberrant mRNA clearance in in vivo genome editing
title_short The position of the target site for engineered nucleases improves the aberrant mRNA clearance in in vivo genome editing
title_sort position of the target site for engineered nucleases improves the aberrant mrna clearance in in vivo genome editing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060192/
https://www.ncbi.nlm.nih.gov/pubmed/32144373
http://dx.doi.org/10.1038/s41598-020-61154-4
work_keys_str_mv AT leejaehoon thepositionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT yusungsook thepositionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT namtaewook thepositionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT rohjaeil thepositionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT jinyoung thepositionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT hanjeongpil thepositionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT chajiyoung thepositionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT kimyoonki thepositionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT yeomsucheong thepositionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT namkitaek thepositionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT leehanwoong thepositionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT leejaehoon positionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT yusungsook positionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT namtaewook positionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT rohjaeil positionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT jinyoung positionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT hanjeongpil positionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT chajiyoung positionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT kimyoonki positionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT yeomsucheong positionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT namkitaek positionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting
AT leehanwoong positionofthetargetsiteforengineerednucleasesimprovestheaberrantmrnaclearanceininvivogenomeediting