CRISPR/Cas9 as an antiviral against Orthopoxviruses using an AAV vector

Siegrist, Cathryn M.; Kinahan, Sean M.; Settecerri, Taylor; Greene, Adrienne C.; Santarpia, Joshua L.

doi:10.1038/s41598-020-76449-9

Title: CRISPR/Cas9 as an antiviral against Orthopoxviruses using an AAV vector

Journal Article · Mon Nov 09 00:00:00 EST 2020 · Scientific Reports

DOI:https://doi.org/10.1038/s41598-020-76449-9· OSTI ID:1725833

Siegrist, Cathryn M. ^[1]; Kinahan, Sean M. ^[2]; Settecerri, Taylor ^[3]; Greene, Adrienne C. ^[4]; Santarpia, Joshua L. ^[2]

Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Univ. of Nebraska Medical Center, Omaha, NE (United States)
Univ. of Nebraska Medical Center, Omaha, NE (United States); National Strategic Research Inst., Albuquerque, NM (United States)
Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
Gritstone Oncology, Inc., Cambridge, MA (United States)

A vaccine for smallpox is no longer administered to the general public, and there is no proven, safe treatment specific to poxvirus infections, leaving people susceptible to infections by smallpox and other zoonotic Orthopoxviruses such as monkeypox. Using vaccinia virus (VACV) as a model organism for other Orthopoxviruses, CRISPR–Cas9 technology was used to target three essential genes that are conserved across the genus, including A17L, E3L, and I2L. Three individual single guide RNAs (sgRNAs) were designed per gene to facilitate redundancy in rendering the genes inactive, thereby reducing the reproduction of the virus. The efficacy of the CRISPR targets was tested by transfecting human embryonic kidney (HEK293) cells with plasmids encoding both SaCas9 and an individual sgRNA. This resulted in a reduction of VACV titer by up to 93.19% per target. Following the verification of CRISPR targets, safe and targeted delivery of the VACV CRISPR antivirals was tested using adeno-associated virus (AAV) as a packaging vector for both SaCas9 and sgRNA. Similarly, AAV delivery of the CRISPR antivirals resulted in a reduction of viral titer by up to 92.97% for an individual target. Overall, we have identified highly specific CRISPR targets that significantly reduce VACV titer as well as an appropriate vector for delivering these CRISPR antiviral components to host cells in vitro.

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Research Organization:: Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

Sponsoring Organization:: USDOE National Nuclear Security Administration (NNSA); USDOD Defense Threat Reduction Agency (DTRA)

Grant/Contract Number:: AC04-94AL85000; NA0003525

OSTI ID:: 1725833

Report Number(s):: SAND-2020-13036J; 692450

Journal Information:: Scientific Reports, Vol. 10, Issue 1; ISSN 2045-2322

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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