Arrayed lentiviral CRISPR screening? Not so fast. Lessons learned from RNAi
Synthetic crRNA and tracrRNA can be used in high-throughput screening applications
Creating patient-specific genome-altered cells for transplant has long been a therapeutic goal in the biomedical research field. In studying HIV infection, two of the biggest hurdles for achieving this goal have been the development of technology appropriate to make precise gene alterations without potential off-target effects in biologically relevant cell types and gene editing reagent delivery that does not rely on viral transduction. By applying the latest CRISPR-Cas9 genetic tools in a novel high-throughput screening format in primary CD4+ cells, Hultquist et al.  have made great strides towards solving these issues and utilized the assay system to identify host dependency factors important in HIV infection and pathogenesis.
The publication describes development of a robust, high-throughput assay system that relies on electroporation of CRISPR-Cas9 ribonucleoproteins (RNPs). Functional CRISPR-Cas9 RNPs can be assembled by complexing synthetic CRISPR-targeting RNA (crRNA), synthetic trans-activating RNA (tracrRNA) and Cas9 protein . Multiple crRNA designs to several genes were synthesized in 96-well format, complexed into CRISPR-Cas9 RNPs and delivered by electroporation. The scalable system allowed for delivery of the CRISPR-Cas9 RNPs to several different blood donor samples allowing for the generation of multiple gene edited cell populations. The experimental approach’s robustness was validated by measurement of phenotype, gene editing percentage and gene knockout. Specifically, CRISPR-Cas9 RNPs were used to efficiently edit and knockout CXCR4 or CCR5 in primary T-cells, and the knockout of each gene was shown to decrease HIV-1 infection. The results suggested that the assay was appropriate for monitoring permissivity determinants of infection, and consistent results across multiple donor cell populations reinforced the effectiveness of the platform.
"The ease of use, high efficiency, broad accessibility, and functional adaptability make this platform truly revolutionary"
Judd Hultquist, University of California
A unique benefit of CRISPR-Cas9 RNPs that utilize synthetic crRNA and tracrRNA was demonstrated: multiplexed gene knockouts can be generated in donor T cells by mixing Cas9 RNPs targeting different genes before electroporation. The CXCR4 and LEDGF genes were simultaneously targeted for gene knockout resulting in ~ 20 % double knockout cells that showed a combined resistance to VSV-G and CXCR4-pseudotyped viruses as effectively as the respective single knockout cells.
The main advantage of this high-throughput editing approach is that it allows for screening in an arrayed format, because one-gene-per-well analysis enables a variety of phenotypic readouts and follow up experiments. The team used assay conditions defined in smaller scale experiments and expanded them to perform a high-throughput CRISPR screen of 45 genes previously reported to be involved in HIV integrase function. Testing three crRNA designs per gene in 96-well format, they monitored gene editing and protein knockout, and correlated these changes with phenotypic assay results revealing alterations of infection rates. Thus, the authors demonstrated the usefulness of an adaptable and scalable arrayed CRISPR screening approach to identify novel host factors for HIV pathogenesis and infection in primary T cells.
When speaking about the use of Dharmacon Edit-R CRISPR Cas9 reagents for this work, the paper’s lead author Judd Hultquist said “This work has opened up a lot of new scientific possibilities for us. The ease of use, high efficiency, broad accessibility, and functional adaptability make this platform truly revolutionary and we have already received a lot of interest from other labs in our fields regarding the protocols and reagents.”
- Hultquist JF, Schumann K, Woo JM, Manganaro L, McGregor MJ, Doudna J, Simon V, Krogan NJ, Marson A. A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells. (2016) Cell Rep., 17(5):1438-1452.
- Kelley ML, Strezoska Ž, He K, Vermeulen A, Smith A. Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing. (2016) J Biotechnol Sep 10;233:74-83. doi:10.1016/j.jbiotec.2016.06.011.
- Pre-designed or custom designs to power rapid, efficient gene editing.
- Configurable expression constructs or DNA-free options for optimal Cas9 expression.
- Chemically synthesized trans-activating CRISPR RNA required for use with synthetic crRNA for fast and straightforward gene editing.
- This webinar focuses on CRISPR-Cas9 background and crRNA design considerations, transfection optimization for maximal editing efficiency, enrichment of cell populations with gene editing events by FACS, clonal selection, and provides a detailed analysis of editing events.
- Explains key factors for arrayed crRNA screening, recommendations for Cas9 nuclease selection and optimization, and the dual CRISPR guide RNA system.