A highly efficient gene disruption strategy reveals lipid co-regulatory networks

Abstract

SUMMARYGene disruption has been drastically facilitated by recent genome editing tools. While the efficiency of gene disruption in cell culture has improved, clone isolation remains routinely performed to obtain fully mutated cells, potentially leading to artifacts due to clonal variances in cellular phenotypes. Here we report GENF, a highly efficient strategy to disrupt genes without isolating clones, which can be multiplexed. We obtained reliable lipidomics datasets from mutant cells generated with GENF, which was impossible when using clones. Through this, we found that an enzyme involved in congenital generalized lipodystrophy regulates glycerophospholipids with specific acyl-chains. We also demonstrate the possibility to dissect complex lipid co-regulatory networks and provide some common mechanisms explaining the adaptations to altered lipid metabolism. GENF is likely to contribute to all experimental setups affected by clonal variability and should be especially useful for -omics approaches.

Ceramide synthase 2 deletion decreases the infectivity of HIV-1

“…These knockouts also were generated by CRISPR/Cas9 technology ( 24 ), but rather than clonal cell lines, they represented selected cell populations in which some, but not all cells, bore double allele knockouts. Nevertheless, recent lipidomic analyses indicated that the CerS5 knockout cells were reduced in short and long chain Cer and SM species, whereas CerS2 knockout cells were reduced in very long Cer, SM, and HexCer species ( 45 ). For our analyses, transfected HeLa cell variants expressing Tat plus either VSV G or HIV-1 Env were subjected to fusion assays with TZM-bl reporter cells as described in Figures 5 and 6 .…”

“…Plasmids were cotransfected into HeLa cells using Lipofectamine 3000 (Thermo Fisher Scientific). At 5 days post-transfection, cells were selected with 6 μg/ml 6-thioguanine for 1 week, and populations so selected were employed in our analyses: lipid analyses of these cells have been described recently ( 45 ). All cells were grown in humidified 5% carbon dioxide air at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum plus 10 mM Hepes (pH 7.3), penicillin, and streptomycin.…”

The sterol transporter STARD3 transports sphingosine at ER-lysosome contact sites

“…Gene disruption strategy used to produce CRISPR Cas9 knock-out cells was adapted from a protocol by T. Harayama (IPMC, Valbonne, France). 57 Empty pX330 and HPRT1-3m13 vectors were a kind gift from T. Harayama. The STARD3 sequence was subcloned into the pX330 vector as described above.…”

Optical Control of Membrane Fluidity Modulates Protein Secretion

“…A plasmid for mammalian cell expression of ACSL4-targeting single-guide RNA (sgRNA) and Cas9 was constructed by annealing the oligonucleotides caccgTCATGGGCTAAATGAATCTG (target sequence in upper case) and aaacCAGATTCATTTAGCCCATGAc and ligating them with Quick Ligase (New England Biolabs) into pX330 plasmid (Addgene #42230, deposited by Feng Zhang) cleaved with FastDigest BpiI (Thermo Scientific). This plasmid (495 ng) was co-transfected with 5 ng of a previously generated mismatched sgRNA expression plasmid (Harayama et al, 2020) to target HPRT1 (target sequence with mismatches in lower cases: gtGCCCTCTGTGTGCTCAA) using Lipofectamine 3000. Five days after transfection, mutant cells were selected with 6 µg/mL 6-thioguanine for one week, which kills cells with a functional HPRT1 gene.…”

“…TGC GCT GCT TT Fold change in transcript levels was calculated using the cycle threshold (CT) comparative method (2 −ddCT ) normalizing to CT values of internal control genes GAPDH.Generation of ACSL4 mutant cellsACSL4 polyclonal HeLa MZ mutant cells were generated with CRISPR-Cas9, using the highly efficient co-targeting strategy GENF(Harayama et al, 2020) (GEne co-targeting with Non-eFficient conditions) as following. A plasmid for mammalian cell expression of ACSL4-targeting single-guide RNA (sgRNA) and Cas9 was constructed by annealing the oligonucleotides caccgTCATGGGCTAAATGAATCTG (target sequence in upper case) and aaacCAGATTCATTTAGCCCATGAc and ligating them with Quick Ligase (New England Biolabs) into pX330 plasmid (Addgene #42230, deposited by Feng Zhang) cleaved with FastDigest BpiI (Thermo Scientific).…”