Abstract
The CRISPR-Cas9 system has been used for genome editing of various organisms. We reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections with a single guide RNA (gRNA) and subsequent viral escape, but complete inactivation of infectious HIV with certain combinations of two gRNAs. The new RNA-guided endonuclease system CRISPR-Cas12a (formerly Cpf1) may provide a more promising tool for genome engineering with increased activity and specificity. We compared Cas12a to the original Cas9 system for inactivation of the integrated HIV DNA genome. Superior antiviral activity is reported for Cas12a, which can achieve full HIV inactivation with only a single gRNA (called crRNA). We propose that the different architecture of Cas9 versus Cas12a endonuclease explains this effect. We also disclose that DNA cleavage by the Cas12a endonuclease and subsequent DNA repair causes mutations with a sequence profile that is distinct from that of Cas9. Both CRISPR systems can induce the typical small deletions around the site of DNA cleavage and subsequent repair, but Cas12a does not induce the pure DNA insertions that are routinely observed for Cas9. Although these typical signatures are apparent in many literature studies, this is the first report that documents these striking differences.
Highlights
The ability to add, remove, or change DNA sequences is essential to studies that investigate how genetics cause certain phenotypic traits
The nonhomologous end joining (NHEJ) DNA repair mechanism of the host cell is responsible for the mutations in the viral genome that facilitated human immunodeficiency virus (HIV) escape
For the crRNA design we used the Benchling CRISPR Guide Design Software and we selected crRNAs that target relatively conserved HIV sequences. This latter property will broaden the therapeutic potential towards other HIV isolates and even distinct HIV subtypes [26], but will restrict the likelihood of viral escape as less sequence variation is usually allowed in conserved HIV domains [27]
Summary
The ability to add, remove, or change DNA sequences is essential to studies that investigate how genetics cause certain phenotypic traits. We and others used CRISPR strategies to target the DNA genome of the human immunodeficiency virus (HIV) [3,4,5,6,7,8,9]. This pathogenic virus causes a persistent infection that can be controlled by antiretroviral drugs, but a cure is never reached. HIV can persist because it deposits a DNA copy of its genome into that of the host cell, the so-called integrated HIV provirus that frustrates cure attempts. Combinations of two gRNAs were subsequently tested and we identified two unique gRNA combinations that trigger full virus inactivation in an infected T cell line: the cure in a bottle [10]
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