Editing the human cytomegalovirus genome with the CRISPR/Cas9 system.

Abstract:

:Human Cytomegalovirus (HCMV) is an opportunistic pathogen that causes substantial disease in neonates and immunocompromised individuals. Reverse genetic analysis of the HCMV genome is a powerful tool to dissect the roles that various viral genes play during infection. However, genetic engineering of HCMV is hampered by both the large size of the HCMV genome and HCMV's slow replication cycle. Currently, most laboratories that genetically engineer HCMV employ Bacterial Artificial Chromosome (BAC) mediated recombineering, which is a relatively lengthy process. We explored an alternative method of producing recombinant HCMV using the CRISPR/Cas9 system. We employed both homologous recombination (HR) and Non-homologous end-joining (NHEJ)-based methods, and find that each approach is capable of efficiently mutating the HCMV genome, with optimal efficiencies of 42% and 81% respectively. Our results suggest that CRISPR-mediated genomic engineering of HCMV is competitive with BAC-mediated recombineering and provide a framework for using CRISPR/Cas9 for mutational analysis of the HCMV genome.

journal_name

Virology

journal_title

Virology

authors

King MW,Munger J

doi

10.1016/j.virol.2019.01.021

subject

Has Abstract

pub_date

2019-03-01 00:00:00

pages

186-194

eissn

0042-6822

issn

1096-0341

pii

S0042-6822(19)30022-4

journal_volume

529

pub_type

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