Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line

Essletzbichler, Patrick; Konopka, Tomasz; Santoro, Federica; Chen, Doris; Gapp, Bianca V.; Královics, Róbert; Brummelkamp, Thijn R.; Nijman, Sebastian M.; Bürckstümmer, Tilmann

doi:10.1101/gr.177220.114

Cited by 244 publications

(225 citation statements)

References 44 publications

(59 reference statements)

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“…HAP1 cells have a haploid karyotype except for a fragment of chromosome 15, which is located in chromosome 19, and these cells also contain a reciprocal translocation between chromosomes 9 and 22 (36,37). None of these features affects the structures of the three genes ATP5G1, ATP5G2, and ATP5G3 encoding the three different precursors of the c-subunit of ATP synthase, which are found, respectively, on the single-copy chromosomes 17, 12, and 2.…”

Section: Resultsmentioning

confidence: 99%

Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase

Ford

Carroll

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

217

241

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The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme's rotor. The c-subunit is produced from three nuclear genes, ATP5G1, ATP5G2, and ATP5G3, encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1, ATP5G2, and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F 1 -catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ 0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP.human mitochondria | ATP synthase | permeability transition pore | subunit c | ATP5G

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Section: Resultsmentioning

confidence: 99%

Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase

Ford

Carroll

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

217

241

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“…In addition to generating gene knockouts, the CombiGEM-CRISPR platform could be used for high-order combinatorial gene activation and repression studies by incorporating gRNAs and deactivated Cas9 variants repurposed as transcriptional and epigenetic regulators (8,9,51,52). This technology could also be used to interrogate the function of large genomic deletions (53) and rearrangements (54,55) with barcoded gRNA pairs. Thus, CombiGEM-CRISPR provides a facile approach to uncover gene and drug combinations that exert desired biological responses, especially for phenotypes that require more than a single perturbation to be manifested due to underlying complex biological networks.…”

Section: Discussionmentioning

confidence: 99%

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

Wong

Choi

Cui

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

218

171

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The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. Here, we created a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translated these hits into effective drug combinations. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (Combi-GEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing. We applied CombiGEM-CRISPR to create a library of 23,409 barcoded dual guide-RNA (gRNA) combinations and then perform a high-throughput pooled screen to identify gene pairs that inhibited ovarian cancer cell growth when they were targeted. We validated the growth-inhibiting effects of specific gene sets, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, via individual assays with CRISPR-Cas-based knockouts and RNA-interference-based knockdowns. We also tested small-molecule drug pairs directed against our pairwise hits and showed that they exerted synergistic antiproliferative effects against ovarian cancer cells. We envision that the CombiGEM-CRISPR platform will be applicable to a broad range of biological settings and will accelerate the systematic identification of genetic combinations and their translation into novel drug combinations that modulate complex human disease phenotypes.CRISPR-Cas | CombiGEM | multifactorial genetics | genetic perturbations | high-throughput screening

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“…First proof-of-principle studies showed that Cas9 can be successfully targeted to endogenous genes in bacteria (12), human pluripotent stem cells (13), as well as in whole organisms such as zebrafish (14), yeast (15), fruit flies (16), mice (17), rats (18) and rabbits (19). In addition, a haploid human cell line named engineered-HAPloid cells has been generated by megabase-scale deletion using CRISPR/Cas9 (20).…”

Section: Introductionmentioning

confidence: 99%

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

et al. 2017

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Abstract. Genomic editing using the CRISPR/Cas9 technology allows selective interference with gene expression. With this method, a multitude of haploid and diploid cells from different organisms have been employed to successfully generate knockouts of genes coding for proteins or small RNAs. Yet, cancer cells exhibiting an aberrant ploidy are considered to be less accessible to CRISPR/Cas9-mediated genomic editing, as amplifications of the targeted gene locus could hamper its effectiveness. Here we examined the suitability of CRISPR/Cas9 to knockout the receptor tyrosine kinase Axl in the human hepatoma cell lines HLF and SNU449. The genomic editing events were validated in two single cell clones each from putative HLF and SNU449 knockout cells (HLF-Axl --1, HLF-Axl --2, SNU449-Axl --1, SNU449-Axl --2). Sequence analysis of respective AXL loci revealed one to six editing events in each individual Axl -clone. The majority of insertions and deletions in the AXL gene at exon 7/8 resulted in a frameshift and thus a premature stop in the coding region.However, one genomic editing event led to an insertion of two amino acids resulting in an altered protein sequence rather than in a frameshift in the AXL locus of the SNU449-Axl --1 cells. Notably, while no Axl protein expression could be detected by immunoblotting in all four cell clones, both expression of total Axl as well as release of soluble Axl into the supernatant was observed by ELISA in incompletely edited SNU449-Axl --1 cells. Importantly, a comparative genomic hybridization array revealed comparable genomic changes in Axl knockout cells as well as in cells expressing Cas9 nickase without guide RNAs in SNU449 and HLF cells, indicating vast alterations in genomic DNA triggered by nickase. Together, these data show that the dynamics of CRISPR/Cas9 may cause incomplete editing events in cancer cell lines, as gene copy numbers vary based on genomic heterogeneity.

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Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line

Cited by 244 publications

References 44 publications

Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase

Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

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