PMID- 36926973
OWN - NLM
STAT- Publisher
LR  - 20231020
IS  - 2165-0497 (Electronic)
IS  - 2165-0497 (Linking)
VI  - 11
IP  - 2
DP  - 2023 Mar 16
TI  - Excision of Integrated Human Herpesvirus 6A Genomes Using CRISPR/Cas9 Technology.
PG  - e0076423
LID - 10.1128/spectrum.00764-23 [doi]
LID - e00764-23
AB  - Human herpesviruses 6A and 6B are betaherpesviruses that can integrate their 
      genomes into the telomeres of latently infected cells. Integration can also occur 
      in germ cells, resulting in individuals who harbor the integrated virus in every 
      cell of their body and can pass it on to their offspring. This condition is 
      termed inherited chromosomally integrated HHV-6 (iciHHV-6) and affects about 1% 
      of the human population. The integrated HHV-6A/B genome can reactivate in 
      iciHHV-6 patients and in rare cases can also cause severe diseases including 
      encephalitis and graft-versus-host disease. Until now, it has remained impossible 
      to prevent virus reactivation or remove the integrated virus genome. Therefore, 
      we developed a system that allows the removal of HHV-6A from the host telomeres 
      using the CRISPR/Cas9 system. We used specific guide RNAs (gRNAs) targeting the 
      direct repeat region at the ends of the viral genome to remove the virus from 
      latently infected cells generated in vitro and iciHHV-6A patient cells. 
      Fluorescence-activated cell sorting (FACS), quantitative PCR (qPCR), and 
      fluorescence in situ hybridization (FISH) analyses revealed that the virus genome 
      was efficiently excised and lost in most cells. Efficient excision was achieved 
      with both constitutive and transient expression of Cas9. In addition, reverse 
      transcription-qPCR (RT-qPCR) revealed that the virus genome did not reactivate 
      upon excision. Taken together, our data show that our CRISPR/Cas9 approach allows 
      efficient removal of the integrated virus genome from host telomeres. IMPORTANCE 
      Human herpesvirus 6 (HHV-6) infects almost all humans and integrates into the 
      telomeres of latently infected cells to persist in the host for life. In 
      addition, HHV-6 can also integrate into the telomeres of germ cells, which 
      results in about 80 million individuals worldwide who carry the virus in every 
      cell of their body and can pass it on to their offspring. In this study, we 
      develop the first system that allows excision of the integrated HHV-6 genome from 
      host telomeres using CRISPR/Cas9 technology. Our data revealed that the 
      integrated HHV-6 genome can be efficiently removed from the telomeres of latently 
      infected cells and cells of patients harboring the virus in their germ line. 
      Virus removal could be achieved with both stable and transient Cas9 expression, 
      without inducing viral reactivation.
FAU - Aimola, Giulia
AU  - Aimola G
AD  - Institut fur Virologie, Freie Universitat Berlin, Berlin, Germany.
FAU - Wight, Darren J
AU  - Wight DJ
AD  - Institut fur Virologie, Freie Universitat Berlin, Berlin, Germany.
FAU - Flamand, Louis
AU  - Flamand L
AUID- ORCID: 0000-0001-5010-4586
AD  - Division of Infectious and Immune Diseases, CHU de Quebec Research Center-Laval 
      University, Quebec, Canada.
AD  - Department of Microbiology, Infectious Disease and Immunology, Faculty of 
      Medicine, Laval University, Quebec, Canada.
FAU - Kaufer, Benedikt B
AU  - Kaufer BB
AUID- ORCID: 0000-0003-1328-2695
AD  - Institut fur Virologie, Freie Universitat Berlin, Berlin, Germany.
AD  - Veterinary Centre for Resistance Research (TZR), Freie Universitat Berlin, 
      Berlin, Germany.
LA  - eng
PT  - Journal Article
DEP - 20230316
PL  - United States
TA  - Microbiol Spectr
JT  - Microbiology spectrum
JID - 101634614
SB  - IM
PMC - PMC10100985
OTO - NOTNLM
OT  - CRISPR/Cas9
OT  - HHV-6
OT  - excision
OT  - gRNAs
OT  - iciHHV-6
OT  - integration
COIS- The authors declare no conflict of interest.
EDAT- 2023/03/18 06:00
MHDA- 2023/03/18 06:00
PMCR- 2023/03/16
CRDT- 2023/03/17 08:00
PHST- 2023/03/17 08:00 [entrez]
PHST- 2023/03/18 06:00 [pubmed]
PHST- 2023/03/18 06:00 [medline]
PHST- 2023/03/16 00:00 [pmc-release]
AID - 00764-23 [pii]
AID - spectrum.00764-23 [pii]
AID - 10.1128/spectrum.00764-23 [doi]
PST - aheadofprint
SO  - Microbiol Spectr. 2023 Mar 16;11(2):e0076423. doi: 10.1128/spectrum.00764-23.