The CRISPR/Cas9 Gene Editing System to Inactivate HIV: Non-Specific Effects

Objectives: The long-term goal of our study is to inactivate the HIV-1 genome integrated into the chromosomes of host cells and eliminate the latent viral reservoir. The clustered, regularly interspersed short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas9) system is able to target and cleave specific sequences of DNA, and enable the cell’s DNA repair system to restore the broken DNA strands. This procedure is error-prone and introduces new oligonucleotide sequences into the DNA.
Methods: HeLa, HeLa-tat-III, and HeLa-tat-III/LTR/d1EGFP cells were seeded in 48-well culture plates 1 day prior to transfection, at a density of 1.5 x 10⁵ cells per well in 1 ml of DME medium with 10% serum, 2 mM L-glutamine, and 1% antibiotics, and incubated at 37 degrees Celsius under 5% CO₂. Alamar Blue was used to measure cell viability. The effect of the CRISPR/Cas9 system on LTR-driven gene expression was measured by employing the p3’BlueLTR-Luc plasmid (AIDSReagent, NIH) as a target, and measuring luciferase activity (Promega). pGS-gRNA encoding the different guide RNAs (gRNAs; 20 bp) and pSpCas9 encoding Cas9, both driven by the human U6 promoter, were synthesized by GenScript.
Results: HeLa-tat-III cells expressed 14-fold higher luciferase activity when transfected with pLTR-Luc compared to HeLa cells, indicating the importance of the HIV transactivator protein, Tat. Control, non-targeted gRNA+Cas9 did not affect Luc expression; however, Cas9 alone (without any gRNA), reduced Luc by 20-fold, revealing a non-specific inhibition of gene expression. gRNA-A and gRNA-B, targeting the HIV promoter (LTR) adapted from published works either had no effect or increased Luc expression. gRNA-C, designed in our laboratory, reduced Luc expression 3-fold in one experiment and slightly in another experiment, whereas gRNA-D had variable effects.
Conclusions: The enhancement of LTR-driven luciferase expression by the CRISPR/ Cas9 system by some gRNAs was unexpected, and contradicts the hypothesis that this system, with gRNA targeted to the LTR, will inhibit the expression of luciferase driven by the LTR promoter. The observation that Cas9 alone without gRNA inhibited gene expression from pLTR-Luc is another non-specific effect. There may competition between the U6 and LTR promoters for cellular transcription factors.