Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput screening platform for the optimization of CRISPR-Cas9 delivery strategies. By exploiting high content image analysis and microcontact printed plates, multi-parametric gene-editing outcome data from hundreds to thousands of isolated cell populations can be screened simultaneously. Employing this platform, we systematically screened four commercially available cationic lipid transfection materials with a range of RNAs encoding the CRISPR-Cas9 system. Analysis of Cas9 expression and editing of a fluorescent mCherry reporter transgene within human embryonic kidney cells was monitored over several days after transfection. Design of experiments analysis enabled rigorous evaluation of delivery materials and RNA concentration conditions. The results of this analysis indicated that the concentration and identity of transfection material have significantly greater effect on gene-editing than ratio or total amount of RNA. Cell subpopulation analysis on microcontact printed plates, further revealed that low cell number and high Cas9 expression, 24 hours after CRISPR-Cas9 delivery, were strong predictors of gene-editing outcomes. These results suggest design principles for the development of materials and transfection strategies with lipid-based materials. This platform could be applied to rapidly optimize materials for gene-editing in a variety of cell/tissue types in order to advance genomic medicine, regenerative biology and drug discovery.
HIV-1 genome integration and capsid assembly are some of the most important steps to viral life cycle. Designed scaffold proteins, zinc finger and ankyrin repeat protein, were characterized as intracellular antiviral agents that can interfere in these steps. Zinc finger protein, 2LTRZFP, plays a vital role in blocking HIV integration. This protein targets the 2-LTR-circle junction of the HIV-1 DNA but it would have no effect on the HIV-infected cells. Another protein, AnkGAG1D4, will then specifically bind to the viral capsid resulting in inhibition of HIV-1 assembly. Since only one particular scaffold protein cannot confer full protection against the virus. Combination of these two proteins became the key to enhance inhibitory function to block HIV-1 replication at early and late infection stage and also help eliminate latent viral reservoirs. The human T cell line modified by using a lentiviral vector bearing 2LTRZFP alone or combination with N-myristoylatedMyr(+)AnkGAG1D4 were protected from viral infection in HIV-1 challenge. Interestingly, the Myr(+)AnkGAG1D4 showed significantly antiviral effect in HIV-1-preinfected SupT1 cells and also possessed a broad antiviral activity against SIV, and SHIV. These results demonstrate that 2LTRZFP and Myr(+)AnkGAG1D4 proteins can be the novel anti-HIV-1 therapeutic agents for human applications.
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