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In March 2016, a very exciting preclinical study was published by a team from Temple University. They demonstrated an important proof-of-concept that a human CD4+ T cell with an integrated HIV genome can be targeted with one or two gRNAs to completely eliminate the viral genome from host cells, with almost no off-target effects to speak of (Kaminsky et al., Science 2016). This is work represents a new therapeutic avenue to cure a pandemic that has affected 79 million patients since 1981.
Although the Temple team focused on a specific HIV-1 quasi-species that had infected a stable cell line in laboratory conditions, the method that they used to build their data model and clear CD4+ T cells of HIV in vitro is likely representative of how this could also be done in patients. Specifically, they used whole genome sequencing to verify the location of the integrated proviral DNA, which not only informed them (and in the future, a researcher or clinician) about what type of HIV strain they were dealing with, but also provided them with the dataset needed to run a complete off-target analysis, that considers the unique genome of the patient (or in this case, cell line). In addition to serving as a gold-standard diagnostic, the whole genome sequencing analysis also serves as the all-important step in ensuring that off-target effects are avoided and that the CRISPR/Cas9 system only targets the viral genome. It also allowed the team to design guides targeting the specific HIV strain. For patients co-infected with more than one type of HIV, presumably guides could be designed against multiple strains using this technique.
Overcoming the current risks of personalized CRISPR
The Temple University team were able to minimize the number and severity of off-target effects their guides exhibited in their study. While off-target effects are sometimes tolerated in basic studies using CRISPR, preclinical and clinical use of CRISPR would necessarily have to minimize or eliminate such effects completely. The Temple University team nonetheless found some off-target effects in their cells, and while these did not appear to harm the cells in a meaningful way, this could potentially harm patient in a clinical setting. A question clinicians will have to ask will be whether or not these minimal off-target hits can be tolerated in patients (IE is it “safe enough”).
Notably, the Temple team used hSpCas9 in their study, and so there is plenty of room to optimise the technique by switching out the Cas9 orthologue to one with fewer off-target effects such as the eSPpCas9 (Slaymaker et al., Science 2015) or SpCas9-HF1(Kleinstiver et al., Nature 2016). To be sure, the presence of any off-targets whatsoever, regardless of the criteria used, suggests that there is more work to be done to create a categorically safe treatment that can be reliably used in patients, or otherwise avoid any off-target effects in cell lines in vitro.
In the meantime, the most important outcome of this paper is a demonstration of the complete excision of the HIV proviral genome using only two gRNAs targeting two sites. This is incredible work.
This article is a revised version of a post originally found on the Desktop Genetics blog. Desktop Genetics is a company building better tools for CRISPR genome editing.
Edward Perello is the founder of Desktop Genetics, a company at the forefront of CRISPR genome editing technology. His team is working to provide researchers with access to state of the art genome engineering capabilities from their computers and create an AI that can predict optimal genome editing solutions in any organism.
Edward is a SynBio LEAP fellow working to get more non-biologists into the field.