Retroelement Insertion in a CRISPR/Cas9 Editing Site in the Early Embryo Intensifies Genetic Mosaicism.
CRISPR
Cas9
embryo
embryonic stem cell
indel depth
mosaicism
sgRNA
Journal
Frontiers in cell and developmental biology
ISSN: 2296-634X
Titre abrégé: Front Cell Dev Biol
Pays: Switzerland
ID NLM: 101630250
Informations de publication
Date de publication:
2019
2019
Historique:
received:
08
08
2019
accepted:
23
10
2019
entrez:
30
11
2019
pubmed:
30
11
2019
medline:
30
11
2019
Statut:
epublish
Résumé
Continued CRISPR/Cas9-mediated editing activity that allows differential and asynchronous modification of alleles in successive cell generations expands allelic complexity. To understand the earliest events during CRISPR/Cas9 editing and the allelic selection among the progeny of subsequent cell divisions, we inspected in detail the genotypes of 4- and 8-cell embryos and embryonic stem cells (ESCs) after microinjection of a CRISPR toolkit into the zygotes. We found a higher editing frequency in 8-cell embryos than in 4-cell embryos, indicating that the CRISPR/Cas9 activity persisted through the 8-cell stage. Analysis of a CRISPR/Cas9 transgenic founder mouse revealed that four different alleles were present in its organs in different combinations and that its germline included three different mutant alleles, as shown by the genotypes of the pups. The indel depth, which measured the extent of indels at the sequence level within single embryos, decreased significantly as the embryos advanced to form ESCs, suggesting that exclusion of fatal indels occurred in the subsequent cell generations. Interestingly, we discovered that the CRISPR sites frequently contained introduced retroelement sequences and that this occurred preferentially with certain classes of retroelements. Therefore, in addition to CRISPR/Cas9's innate mechanism of separate, differential enzymatic modifications of alleles, the frequent retroelement insertions that occur in early mouse embryos during CRISPR/Cas9 editing further expand the allelic diversity and mosaicism in the resulting transgenic founders.
Identifiants
pubmed: 31781562
doi: 10.3389/fcell.2019.00273
pmc: PMC6857330
doi:
Types de publication
Journal Article
Langues
eng
Pagination
273Informations de copyright
Copyright © 2019 Jeon, Park, Min, Chung, Kim and Kang.
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