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The human origin recognition complex (ORC) is essential for pre-RC assembly, mitosis and maintenance of nuclear structure

Citation

Chou, Hsiang-Chen et al. (2021), The human origin recognition complex (ORC) is essential for pre-RC assembly, mitosis and maintenance of nuclear structure, Dryad, Dataset, https://doi.org/10.5061/dryad.59zw3r25f

Abstract

The origin recognition complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre-RC complexes at origins of DNA replication. Here, using tiling-sgRNA CRISPR screens, we report that each subunit of ORC and CDC6 is essential in human cells. Using an auxin-inducible degradation system, we created stable cell lines capable of ablating ORC2 rapidly, revealing multiple cell division cycle phenotypes. The primary defects in the absence of ORC2 were cells encountering difficulty in initiating DNA replication or progressing through the cell division cycle due to reduced MCM2-7 loading onto chromatin in G1 phase. The nuclei of ORC2-deficient cells were also large, with decompacted heterochromatin. Some ORC2-deficient cells that completed DNA replication entered into, but never exited mitosis. ORC1 knockout cells also demonstrated extremely slow cell proliferation and abnormal cell and nuclear morphology. Thus, ORC proteins and CDC6 are indispensable for normal cellular proliferation and contribute to nuclear organization.

Methods

  1. Tiling-sgRNA CRISPR Screen - CRISPR-based negative selection screenings using sgRNA libraries targeting proteins ORC1-6, CDC6 as well as positive and negative controls, were performed in stable Cas9 expressing HCT116 and RPE1 cell lines. The screens were performed as previously described (Lu et al., 2018; Miles et al., 2016; Shi et al., 2015). To quantify the sgRNA abundance of initial and final time-points, the sgRNA cassette was PCR amplified from genomic DNA using Amplitaq Gold DNA Polymerase (Invitrogen, 4311820) and primers (F2: TCTTGTGGAAAGGACGAAACACCG; R2: TCTACTATTCTTTCCCCTGCACTGT).  The resulting DNA fragment (~ 242 bp) was gel purified. In a 2nd PCR reaction illumina-compatible P7 and custom stacked barcodes (Supplement Table 2_BClist) including the standard illumina P5 forward primer were introduced into samples by PCR amplification and gel purified for the final product (~180-200 bp). The final product was quantified by Agilent Bioanalyzer DNA High-sensitivity Assay (Agilent 5067-4626) and pooled together in equal molar ratio and analyzed by NGS. Illumina libraries were either sequenced with a 76 cycle NextSeq 500/550 kit by single-end sequencing using NextSeq mid-output. The quantification of guides was done using a strict exact match to the forward primer, sample barcode, and guide sequence (Supplement Table1_guides).
     
  2. CNV analysis by SMASH - Approximately 500 ng cell genomic DNA was enzymatically fragmented using dsDNA fragmentase (NEB, M0348L). Following end repair, fragments were joined to create chimeric fragments of DNA suitable for creating NGS libraries (300-700bp). The fragment size selection was done with Agencourt AMPure XP beads (Beckman Coulter, Cat. No. A63881). Illumina-compatible NEBNext Multiplex Dual Index Primer Pairs and adapters (New England Biolabs, Cat. No. E6440S) were ligated to the selected chimeric DNA fragments. These barcoded DNA fragments were then sequenced using an Illumina 300cycle MiSeqv2 kit on a MiSeq platform. The SMASH analysis pipeline searches for Maximal Unique Matches (MUMs) to the human genome in all read pairs using a suffix array.  These MUMs were then filtered to exclude short matches below 20bp, matches with less than 4bp of excess unique sequence, and matches on read 2 that are within 1000 bases of the genome coordinate of matches from read 1. The resulting 3-4 on average kept matches per read pair are then added to pre-computed empirically sized bins spanning the genome to generate a raw copy number profile. Regions with identical copy are expected to yield similar bin counts using these empirical bins. This profile is then corrected to remove GC content effects by normalizing counts based on LOWESS smoothing of count vs. GC content data in each bin. Final copy number profiles are normalized so that the autosome has an average copy number of 2. Plots were generated with G-Graph (MUMdex software. MUMdex Genome Alignment Anal. Softw. https://mumdex.com/) (Andrews et al., 2016) .
     
  3. ONT Nanopore Long-Read Sequencing - High molecular weight DNA was isolated using the MagAttract kit (Qiagen # 67563). The quality of the DNA from the was assessed on femtopulse (Agilent) to ensure DNA fragments were >40kb on average DNA was sheared to 50kb via Megarupter (diagenode).  After shearing, the DNA was size selected with a SRE kit (Circulomics) to reduce the fragments <20kb.  After size selection, the DNA under when a-tailing and damage repair followed by ligation to sequencing specific adapters. The ½ prepared library was mixed with library loading beads and loaded on to a PROM-0002 flow-cell and was allowed to sequence for 24 hours.  After 24 hours the flow-cell was treated with DNase to remove stalled DNA followed by a buffer flush. The second ½ of the library was then loaded and allowed to sequencing for 36 hours.  The DNA was base called via Guppy 3.2 in High accuracy mode. Long reads were aligned to the reference human genome using NGMLR (https://github.com/philres/ngmlr) and structural variants were identified using Sniffles (https://github.com/fritzsedlazeck/Sniffles) (Sedlazeck et al., 2018) . The alignments and structural variants were then visualized using IGV (https://igv.org/)

Usage Notes

  1. Tiling_sgRNA_CRISPR_screen.tar.gz
    • ATCACG.fastq.gz    initial time point for HCT116 cell line Replicate1
    • TTAGGC.fastq.gz    initial time point for HCT116 cell line Replicate2
    • TGACCA.fastq.gz    final time point for HCT116 cell line Replicate1
    • GGCTAC.fastq.gz    final time point for HCT116 cell line Replicate2
    • CTTGTA.fastq.gz    initial time point for RPE1 cell line
    • GAGTGG.fastq.gz    final time point for RPE2 cell line
    • HCT116_rep1.csv - LFC values
    • HCT116_rep2.csv - LFC values
    • RPE1.csv - LFC values
    • ReadMe.txt file 
       
  2. SMASH.zip
    • HCT116p53WT folder
      • HCT116p53WT_S6_R1_001.fastq.gz 
      • HCT116p53WT_S6_R2_001.fastq.gz
      • All other file with extensions .ps, .pdf, .txt are outputs of analysis using SMASH
    • HCT116p53null folder
      • HCT116p53null_S2_R1_001.fastq.gz
      • HCT116p53null_S2_R2_001.fastq.gz
      • All other file with extensions .ps, .pdf, .txt are outputs of analysis using SMASH
    • ORC1null folder
      • ORC1null_S3_R1_001.fastq.gz
      • ORC1null_S3_R2_001.fastq.gz
      • All other file with extensions .ps, .pdf, .txt are outputs of analysis using SMASH
    • ORC2null folder
      • ORC2null_S4_R1_001.fastq.gz
      • ORC2null_S4_R2_001.fastq.gz
      • All other file with extensions .ps, .pdf, .txt are outputs of analysis using SMASH
    • ReadMe.txt
       
  3. ORC2null_ONT.zip
    • Alignments Folder - Alignment files
      • ORC2null.sort.bam.bai
      • ORC2null.sort.bam
    • SV_inference Folder - 
      • ORC2_CRISPR_sniffles.vcf - Analysis with Sniffles v1.0.11
      • ORC2_CRISPR_pbsv.vcf - Analysis with PBSV v2.2.0
    • ReadMe.txt

Funding

National Cancer Institute, Award: P01-CA13106

Cancer Center Support Grant, Award: P50-CA045508

Cancer Center Support Grant, Award: P50-CA045508