Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Mar:529:186-194.
doi: 10.1016/j.virol.2019.01.021. Epub 2019 Jan 26.

Editing the human cytomegalovirus genome with the CRISPR/Cas9 system

Melvin W King  1 , Joshua Munger  2
Affiliations

Editing the human cytomegalovirus genome with the CRISPR/Cas9 system

Melvin W King et al. Virology. 2019 Mar.

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.

Keywords: CRISPR; Cas9; Cytomegalovirus; HCMV; Recombineering.

PubMed Disclaimer

Figures

Figure 1:
Figure 1:. Homology-directed recombineering of the HCMV genome in fibroblasts.
(a) Schematic of Cas9 _targeting and GFP incorporation. Homology arms are 750bp. (b) Timeline of cell treatment, infection, transfection and viral isolation over 120 hours. Per well, 10ul of HR template (10ug of GFP-containing HR template in 30uL) was transfected as a linearized plasmid either 24 hours pre- or post-infection. (c) Viral recombination efficiencies. Where indicated, cells were treated with 10ug/mL blasticidin from days 2–5. I-> E denotes infection, then electroporation. E -> I denotes electroporation, then infection.
Figure 2:
Figure 2:. High efficiency CRISPR-mediated INDEL inactivation in the HCMV genome.
(a) Schematic of _targeting viral GFP function using CRISPR/Cas9 editing in primary fibroblasts. 24 hours post-infection, 10uL of 10uM ssODN solution was transfected per well. Where indicated, cells were treated with 10uM SCR7 from days 1–5 (b) GFP KO efficiencies were calculated by plaque assay at 10dpi.
Figure 3:
Figure 3:. Optimization of homology-directed recombineering of the HCMV genome in fibroblasts.
(a) Schematic for repairing a 21-base pair deletion in viral GFP using CRISPR/Cas9 Homologous Recombination. HR template volumes given represent ul from a solution containing 10ug of GFP-containing HR template in 30uL. (b) Recombination efficiencies for repairing GFP function using a small ssODN repair template, counted at 10 days post infection. Volumes given represent ul of a 10 uM oligonucleotide-containing solution. (c) Recombination efficiencies of optimized homologous recombination/insertion of GFP-BSD into the US7 locus. HR template volumes given represent ul from a solution containing 10ug of GFP-containing HR template in 30uL
Figure 4:
Figure 4:
Timeline and Methodological Comparison of BAC recombineering and CRISPR-mediated homologous recombination.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Azevedo LS, Pierrotti LC, Abdala E, Costa SF, Strabelli TMV, Campos SV, Ramos JF, Latif AZA, Litvinov N, Maluf NZ, Caiaffa Filho HH, Pannuti CS, Lopes MH, dos Santos VA, Linardi C. da C.G., Yasuda MAS, Marques H.H. de S., 2015. Cytomegalovirus infection in transplant recipients. Clin. São Paulo, Brazil) 70, 515–23. 10.6061/clinics/2015(07)09 - DOI - PMC - PubMed
    1. Biron KK, Fyfe JA, Stanat SC, Leslie LK, Sorrell JB, Lambe CU, Coen DM, 1986. A human cytomegalovirus mutant resistant to the nucleoside analog 9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl}guanine (BW B759U) induces reduced levels of BW B759U triphosphate. Proc. Natl. Acad. Sci. U. S. A 83, 8769–8773. 10.1073/pnas.83.22.8769 - DOI - PMC - PubMed
    1. Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kühn R, 2015. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol 33, 543–548. 10.1038/nbt.3198 - DOI - PubMed
    1. Damato EG, Winnen CW, 2002. Cytomegalovirus Infection: Perinatal Implications. J. Obstet. Gynecol. Neonatal Nurs 31, 86–92. - PubMed
    1. Janoly-Dumenil A, Rouvet I, Bleyzac N, Morfin F, Zabot MT, Tod M, 2012. A pharmacodynamic model of ganciclovir antiviral effect and toxicity for lymphoblastoid cells suggests a new dosing regimen to treat cytomegalovirus infection. Antimicrob. Agents Chemother 56, 3732–3738. 10.1128/AAC.06423-11 - DOI - PMC - PubMed

Publication types

  NODES
Note 2
twitter 2