This study presents a comprehensive benchmarking of human-derived transcription activation domains (hTADs) for use in CRISPR-based artificial transcription factors (ATFs), aiming to circumvent the immunogenicity associated with viral TADs like VP64 and VPR. The authors systematically compared eight hTADs—CITED1, CITED2, MYB, KLF7, CSRNP1, NFZ, MSN, and p65HSF1—fused to dCas9. While several hTADs surpassed VP64 in activating a synthetic EGFP reporter, none outperformed VPR at endogenous gene targets.
To enhance efficacy, the authors engineered 16 pairwise combinations of the four most promising hTADs (MSN, NFZ, CITED2, and p65HSF1) and tested them across multiple endogenous loci (e.g., HBG, TTN, IL1B, ASCL1, NEUROD1). Notably, combinations such as NFZ-p65HSF1 (NP), CITED2-MSN (CM), and CITED2-p65HSF1 (CP) matched or exceeded VPR in activation potency, while maintaining a smaller size advantageous for gene delivery. These hTAD fusions performed robustly across HEK293T, HeLa, human embryonic stem cells, and even in yeast.
Further experiments demonstrated that NP, in particular, retained strong activation capabilities when fused to the compact dCasMINI protein, enabling the construction of highly compact and efficient CRISPR activators. Transcriptome-wide RNA-seq revealed that NP activated target loci with high specificity, similar to VPR, but with potentially reduced immunogenicity due to its human origin.
This work provides the first direct comparison of recently identified hTADs in a unified framework, offering new compact and effective modules for synthetic gene regulation. The study highlights NP as a leading candidate for future CRISPRa applications in therapeutic or multiplexed settings, combining efficacy, compactness, and reduced immunogenic risk.
This study engineered human-derived transcriptional activators that match viral benchmarks like VPR, offering compact, efficient, and potentially less immunogenic CRISPR tools for precise gene activation.
