Sangeeta Kumari, M.S., Subha Philip, Ph.D., Diana Douglas, B.S., Jarkko Huuskonen, Ph.D., Weiguo Shu, Ph.D.
Developing technologies for the targeted disruption of gene expression will provide powerful tools for studying gene function. To date, various methods for achieving loss-of-function (LOF) outcomes have been developed, including approaches employing RNA interference (RNAi) and CRISPR (clustered regularly interspaced short palindrome repeats)/Cas9-mediated gene perturbation.1 In mammalian cells, RNAi is the predominant method for programmed knockdown of mRNAs, but its utility is limited by confounding off-target effects.2 The RNA-guided CRISPR-associated nuclease Cas9 provides an effective means of introducing targeted LOF mutations in the genome. Cas9 can be programmed to induce DNA double strand breaks (DSBs) at specific genomic loci through guide RNAs (gRNA), which when targeted to coding regions of genes can create frame shift indel mutations resulting in a LOF allele.3 In addition to the use of the nuclease activity of Cas9, the CRISPR-Cas9 technology can also be repurposed as a sequence-specific, non-mutagenic gene regulation tool. Coupling of the engineered nuclease-deficient Cas9 (dCas9) to a transcriptional repressor domain can robustly silence expression of endogenous genes with high specificity, resulting in ‘CRISPR interference’ (CRISPRi).4,5 Here we report the creation of Cas9-expressing HEK-293 and CRISPRi A549 cell lines, in which the Cas9 or KRAB-dCas9 expression cassette was integrated into AAVS1 safe harbor locus. The integration of knock-in allele was confirmed at the genomic and translational levels in both cell lines. When gRNAs targeting p53 and RFP genes were delivered into HEK-293 Cas9 cells, DNA double-stranded breaks at intended sites were detected using T7E1 assay and Sanger sequencing, and the expression of p53 and RFP proteins was significantly disrupted. In A549 CRISPRi cells, gRNAs targeting p53 and SETD9 promoter regions repressed p53 and SETD9 gene transcription approximately 75% and 65%, respectively. In p53 gRNA expressing virus infected A549 CRISPRi single cell clones, approximately 95% p53 transcription repression was detected. Taken together, our data suggest that these CRISPR tool cell lines are valuable tools that greatly simplify the study of human gene function and provide potential applications for precise gene knockout and knockdown in human cells.
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