Cargando…

Precise and broad scope genome editing based on high-specificity Cas9 nickases

RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking R...

Descripción completa

Detalles Bibliográficos
Autores principales: Wang, Qian, Liu, Jin, Janssen, Josephine M, Le Bouteiller, Marie, Frock, Richard L, Gonçalves, Manuel A F V
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826261/
https://www.ncbi.nlm.nih.gov/pubmed/33398349
http://dx.doi.org/10.1093/nar/gkaa1236
_version_ 1783640497803231232
author Wang, Qian
Liu, Jin
Janssen, Josephine M
Le Bouteiller, Marie
Frock, Richard L
Gonçalves, Manuel A F V
author_facet Wang, Qian
Liu, Jin
Janssen, Josephine M
Le Bouteiller, Marie
Frock, Richard L
Gonçalves, Manuel A F V
author_sort Wang, Qian
collection PubMed
description RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9(D10A) variants whose precision is superior to that of the commonly used Cas9(D10A) nickase. Dual nicking RGNs based on a selected group of these Cas9(D10A) variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by ‘tiptoeing’ over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.
format Online
Article
Text
id pubmed-7826261
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-78262612021-01-27 Precise and broad scope genome editing based on high-specificity Cas9 nickases Wang, Qian Liu, Jin Janssen, Josephine M Le Bouteiller, Marie Frock, Richard L Gonçalves, Manuel A F V Nucleic Acids Res Synthetic Biology and Bioengineering RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9(D10A) variants whose precision is superior to that of the commonly used Cas9(D10A) nickase. Dual nicking RGNs based on a selected group of these Cas9(D10A) variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by ‘tiptoeing’ over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount. Oxford University Press 2021-01-04 /pmc/articles/PMC7826261/ /pubmed/33398349 http://dx.doi.org/10.1093/nar/gkaa1236 Text en © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Synthetic Biology and Bioengineering
Wang, Qian
Liu, Jin
Janssen, Josephine M
Le Bouteiller, Marie
Frock, Richard L
Gonçalves, Manuel A F V
Precise and broad scope genome editing based on high-specificity Cas9 nickases
title Precise and broad scope genome editing based on high-specificity Cas9 nickases
title_full Precise and broad scope genome editing based on high-specificity Cas9 nickases
title_fullStr Precise and broad scope genome editing based on high-specificity Cas9 nickases
title_full_unstemmed Precise and broad scope genome editing based on high-specificity Cas9 nickases
title_short Precise and broad scope genome editing based on high-specificity Cas9 nickases
title_sort precise and broad scope genome editing based on high-specificity cas9 nickases
topic Synthetic Biology and Bioengineering
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826261/
https://www.ncbi.nlm.nih.gov/pubmed/33398349
http://dx.doi.org/10.1093/nar/gkaa1236
work_keys_str_mv AT wangqian preciseandbroadscopegenomeeditingbasedonhighspecificitycas9nickases
AT liujin preciseandbroadscopegenomeeditingbasedonhighspecificitycas9nickases
AT janssenjosephinem preciseandbroadscopegenomeeditingbasedonhighspecificitycas9nickases
AT lebouteillermarie preciseandbroadscopegenomeeditingbasedonhighspecificitycas9nickases
AT frockrichardl preciseandbroadscopegenomeeditingbasedonhighspecificitycas9nickases
AT goncalvesmanuelafv preciseandbroadscopegenomeeditingbasedonhighspecificitycas9nickases