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PAF53 is essential in mammalian cells: CRISPR/Cas9 fails to eliminate PAF53 expression.
Author(s) -
Rothblum Lawrence,
Chang Eugenie,
Rothblum Katrina
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.597.4
Subject(s) - biology , crispr , gene , transfection , cas9 , genetics , ribosome biogenesis , plasmid , puromycin , microbiology and biotechnology , ribosome , protein biosynthesis , rna
When mammalian cells are nutrient and/or growth factor deprived, exposed to inhibitors of protein synthesis, stressed by heat shock or grown to confluence, rDNA transcription is essentially shut off. Various mechanisms are available to accomplish this downshift in ribosome biogenesis. Muramatsu's laboratory first demonstrated that mammalian PAF53 was essential for specific rDNA transcription and that PAF53 levels were regulated in response to growth factors While S. cerevisae A49, the homologue of PAF53, is not essential for viability, deletion of yA49 results in colonies that grow at 6% of the wild type rate at 25C. This included the genes encoding Rrn3, PAF49 and PAF53. Experiments described by Wang et al. identified PAF53 as a gene “ essential for optimal proliferation “. However, they did not discriminate genes essential for viability. Hence, in order to resolve this question, we designed a series of experiments to determine if PAF53 was essential for cell survival. We set out to delete the gene product from mammalian cells using CRISPR/CAS9 technology. Methods We designed several small guide RNAs (sgRNA) for human PAF53 and cloned them into lentiCRISPR v2. Human 293 cells were transfected with the viral DNA. In some experiments, the cells were cotransfected in parallel with plasmids encoding tagged forms of mouse PAF53. After treating the transfected cells with puromycin (to select for the lentiCRISPR backbone), cells were cloned and analyzed by western blots for PAF53 expression. Genomic DNA was amplified across the “CRISPRd” exon, cloned and sequenced to identify mutated PAF53 genes. Results We obtained cell lines in which the endogenous PAF53 gene was “knocked out” only when we rescued with FLAF‐PAF53. DNA sequencing demonstrated that in the absence of ectopic PAF53 expression, cells demonstrated unique means of surviving; including recombination or the utilization of alternative reading frames. Conclusions PAF53 is an essential protein. We did not obtain any clones that did not express the protein. PAF53 expression requires both genes. We never observed a clone in which one gene is expressed, unless there was also ectopic expression In the absence of ectopic gene expression, the gene products of both endogenous genes were expressed, irrespective of whether they were partially mutant proteins or not.