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Strategies to reduce genetic mosaicism following CRISPR-mediated genome edition in bovine embryos

Published on 2019in Scientific Reports4.01
· DOI :10.1038/s41598-019-51366-8
Abstract
Genetic mosaicism is the presence of more than two alleles on an individual and it is commonly observed following CRISPR microinjection of zygotes. This phenomenon appears when DNA replication precedes CRISPR-mediated genome edition and it is undesirable because it reduces greatly the odds for direct KO generation by randomly generated indels. In this study, we have developed alternative protocols to reduce mosaicism rates following CRISPR-mediated genome edition in bovine. In a preliminary study we observed by EdU incorporation that DNA replication has already occurred at the conventional microinjection time (20 hpi). Aiming to reduce mosaicism appearance, we have developed three alternative microinjection protocols: early zygote microinjection (10 hpi RNA) or oocyte microinjection before fertilization with either RNA or Ribonucleoprotein delivery (0 hpi RNA or 0 hpi RNP). All three alternative microinjection protocols resulted in similar blastocyst and genome edition rates compared to the conventional 20 hpi group, whereas mosaicism rates were significantly reduced in all early delivery groups (~10–30% of edited embryos being mosaic depending on the loci) compared to conventional 20 hpi microinjection (100% mosaicism rate). These strategies constitute an efficient way to reduce the number of indels, increasing the odds for direct KO generation.
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#1Marcela Vilarino (UC Davis: University of California, Davis)H-Index: 4
#2Sheikh Tamir Rashid (Stanford University)H-Index: 5
Last.Santiago S. Diab (UC Davis: University of California, Davis)H-Index: 10
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#1Hong Ma (Oregon National Primate Research Center)H-Index: 27
#2Nuria Marti-Gutierrez (OHSU: Oregon Health & Science University)H-Index: 1
Last.Riffat Ahmed (OHSU: Oregon Health & Science University)H-Index: 10
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#1Xuemei Zhang (XJU: Xinjiang University)H-Index: 6
#2Wenrong Li (XJU: Xinjiang University)H-Index: 6
Last.Mingjun Liu (XJU: Xinjiang University)H-Index: 3
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#1Taichi Noda (Osaka University)H-Index: 8
#2Asami Oji (Osaka University)H-Index: 7
Last.Masahito Ikawa (Osaka University)H-Index: 66
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