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G. Dong et al.
We identified the tumor suppressor function of PRDM1 in NK cells by knocking down PRDM1 by shRNA transduc- tion.6 The recent development of CRISPR/Cas9 technology provides a powerful and versatile approach to edit the genome, thereby facilitating our understanding of the func- tional alterations induced by specific genetic alterations. Gene inactivation by methylation or mutation can be sim- ulated by introducing small out-of-frame indels or knock- ing-in mutations. We were able to successfully KO selected tumor suppressor genes identified in ENKTCL, including mono-allelic and bi-allelic deletions. KO experiments rely on non-homologous end joining mechanisms and are very
robust, but in the absence of a readily selectable marker, we have to rely on cloning to select for modified cells. This is a long and demanding process and may introduce a selection bias. Therefore, we adopted another approach to knock-in a fluorescent protein gene to disrupt the PRDM1 locus while introducing a marker for selection. We were able to knock-in a GFP or DsRed gene into the PRDM1 locus through HDR so that mono- or bi-allelic KO cells could be identified by single or double fluorescence, respectively. This approach shortened the experimental time and gener- ated a bulk population of PRDM1-/- cells to complement and validate the observations from the cloning approach.
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haematologica | 2021; 106(9)