Insights into the mechanisms underlying the inactivation of HIV-1 proviruses by CRISPR/Cas.

DNA editing using CRISPR/Cas has emerged as a potential treatment for diseases caused by pathogenic human DNA viruses. One potential target is HIV-1, which replicates via a chromosomally integrated DNA provirus. While CRISPR/Cas can protect T cells from de novo HIV-1 infection, HIV-1 frequently becomes resistant due to mutations in the chosen single guide RNA (sgRNA) target site. To address this problem, we asked whether an sgRNA targeted to a conserved, functionally critical HIV-1 sequence might prevent the selection of escape mutants. We report that two sgRNAs specific for the HIV-1 transactivation response (TAR) element produce opposite results: the TAR2 sgRNA rapidly selects for mutants that retain TAR function, but are no longer inhibited by Cas9, while the TAR1 sgRNA fails to select any replication competent TAR mutants, most probably because it is targeted to a region of TAR that is disrupted by even minor mutations.

Optimization of a multiplex CRISPR/Cas system for use as an antiviral therapeutic.

RNA-guided endonucleases or CRISPR/Cas systems have been widely employed for gene engineering/DNA editing applications, and have recently been used against a variety of dsDNA viruses as a potential therapeutic. However, in vivo delivery to specific tissue reservoirs using adeno-associated virus (AAV) vectors is problematic due to the large coding requirement for the principal effector commonly used in these applications, Streptococcus pyogenes (Spy) Cas9. Here we describe design of a minimal CRISPR/Cas system that is capable of multiplexing and can be packaged into a single AAV vector. This system consists of the small Type II Cas9 protein from Staphylococcus aureus (Sau) driven by a truncated CMV promoter/enhancer, and flanked 3' by a poly(A) addition signal, as well as two sgRNA expression cassettes driven by either U6 or ∼70-bp tRNA-derived Pol III promoters. Specific protocols for construction of these AAV vector scaffolds, shuttle cloning of their contents into AAV and lentiviral backbones, and a quantitative luciferase assay capable of screening for optimal sgRNAs, are detailed. These protocols can facilitate construction of AAV vectors that have optimal multiplexed sgRNA expression and function. These will have potential utility in multiplex applications, including in antiviral therapy in tissues chronically infected with a pathogenic DNA virus.

Expression of CRISPR/Cas single guide RNAs using small tRNA promoters.

The in vivo application of CRISPR/Cas-based DNA editing technology will require the development of efficient delivery methods that likely will be dependent on adeno-associated virus (AAV)-based viral vectors. However, AAV vectors have only a modest, ∼4.7-kb packaging capacity, which will necessitate the identification and characterization of highly active Cas9 proteins that are substantially smaller than the prototypic Streptococcus pyogenes Cas9 protein, which covers ∼4.2 kb of coding sequence, as well as the development of single guide RNA (sgRNA) expression cassettes substantially smaller than the current ∼360 bp size. Here, we report that small, ∼70-bp tRNA promoters can be used to express high levels of tRNA:sgRNA fusion transcripts that are efficiently and precisely cleaved by endogenous tRNase Z to release fully functional sgRNAs. Importantly, cells stably expressing functional tRNA:sgRNA precursors did not show a detectable change in the level of endogenous tRNA expression. This novel sgRNA expression strategy should greatly facilitate the construction of effective AAV-based Cas9/sgRNA vectors for future in vivo use.