Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit

Engineering of clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) system has enabled versatile applications of CRISPR beyond targeted DNA cleavage. Combination of nuclease-deactivated Cas9 (dCas9) and transcriptional effector domains allows activation (CRISPRa) or repression (CRISPRi) of target loci. To demonstrate the effectiveness of the CRISPR-mediated transcriptional regulation in chickens, three CRISPRa (VP64, VPR, and p300) and three CRISPRi (dCas9, dCas9-KRAB, and dCas9-KRAB-MeCP2) systems were tested in chicken DF-1 cells. By introducing guide RNAs (gRNAs) targeting near the transcription start site (TSS) of each gene in CRISPRa and CRISPRi effector domain-expressing chicken DF-1 cell lines, significant gene upregulation was induced in dCas9-VPR and dCas9-VP64 cells, while significant downregulation was observed with dCas9 and dCas9-KRAB. We further investigated the effect of gRNA positions across TSS and discovered that the location of gRNA is an important factor for targeted gene regulation. RNA sequencing analysis of IRF7 CRISPRa and CRISPRi- DF-1 cells revealed the specificity of CRISPRa and CRISPRi-based targeted transcriptional regulation with minimal off-target effects. These findings suggest that the CRISPRa and CRISPRi toolkits are an effective and adaptable platform for studying the chicken genome by targeted transcriptional modulation.

Automated summary

The engineering of CRISPR and Cas9 system has allowed for diverse applications beyond DNA cleavage. By combining nuclease-deactivated Cas9 with transcriptional effector domains, gene activation (CRISPRa) or repression (CRISPRi) can be achieved. In this study, the effectiveness of CRISPR-mediated transcriptional regulation was demonstrated in chicken DF-1 cells using various CRISPRa and CRISPRi systems, resulting in significant gene upregulation and downregulation. It was also found that the location of guide RNAs plays a crucial role in targeted gene regulation. RNA sequencing analysis confirmed the specificity and minimal off-target effects of CRISPRa and CRISPRi-based transcriptional modulation in chicken cells.