A simple method using CRISPR-Cas9 to knock-out genes in murine cancerous cell lines

Abstract

CRISPR-Cas9 system can be used to generate knock-out cancer cell lines. An insertion or deletion induced by a single guide RNA (gRNA) is often used to generate knock-out cells, however, some cells express the target gene by skipping the disrupted exon, or by using a splicing variant, thus losing the target exon. To overcome this unexpected expression of the target gene, almost the entire gene can be swapped with a selection marker. However, it is time-consuming to create a targeting vector which contains 5′ and 3′ homology arms flanked by a selection marker. Here, we developed a simple and easy method called SUCCESS (Single-strand oligodeoxynucleotides, Universal Cassette, and CRISPR/Cas9 produce Easy Simple knock-out System), to knock-out a target gene without constructing a targeting vector. Our method removed the targeted large genomic region by using two pX330 plasmids encoding Cas9 and gRNA, two 80mer single strand oligodeoxynucleotides (ssODN), and a blunt-ended universal selection maker sequence in B16F10 murine cancer cell and ID8 murine ovarian cancer cell. SUCCESS generated knock-out clones in two murine cancer cell lines by homozygous deletion of the target genomic region, and without constructing targeting vectors. Thus, our method can be widely applied to generate homozygous knock-out cell lines, as well as knock-in cell lines.

N-Acetylglucosamine Sensing and Metabolic Engineering for Attenuating Human and Plant Pathogens

“…Since the emergence of knockout strategies, they have been the most commonly and widely used method for establishing an understanding of human pathogens. The emergence of more efficient knockout generation technologies, like CRISPR, has contributed considerably to the genetic engineering of pathogens [ 67 ]. C. albicans was among the first human pathogens in which the GlcNAc metabolic pathway was thoroughly studied, and Bhattacharya et al [ 68 ] first reported that the presence of GlcNAc is essential for the induction of GlcNAc kinase.…”

Fast-Track and Integration-Free Method of Genome Editing by CRISPR/Cas9 in Murine Pluripotent Stem Cells

“…Additionally, the PCR experiment with PSCs 10 days post-transfection does not reveal any detectable amplicon of transgene Cas9 (Figure 3). The most laborious step in viral delivery is to expand mutant cell pools into single clones (e.g., 96-well plate), which, depending on protocols, requires additional 2-3 weeks or more (Lino et al, 2018;Giuliano et al, 2019;Ishibashi et al, 2020;Lu et al, 2020). Some protocols also require both puromycin-and FACS-based selection, although this technique is praiseworthy since only cells with the highest transduction efficiency are selected (Lino et al, 2018;Giuliano et al, 2019;Ishibashi et al, 2020;Lu et al, 2020), yet these require sophisticated lab setup with flow cytometry and expertise, and are also very time-consuming and laborious.…”

Function of cofactor Akirin2 in the regulation of gene expression in model human Caucasian neutrophil-like HL60 cells

“…For RNAseq analysis of HL60 cells, we compared akirin2 KO with WT cells as previously shown in CRISPR-Cas9 experiments (i.e. [30]) (Supplementary Data S2). RNA was isolated from WT and akirin2 KO HL60 cells (three Upper plots show the thermogram and lower plots show the interaction isotherm.…”