DOI: 10.5061/dryad.59zw3r25f
title: The human origin recognition complex (ORC) is essential for pre-RC assembly, mitosis and maintenance of nuclear structure

description: 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.
description: 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
 2<sup>nd</sup> 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).<br>   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) .<br>
   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 &gt;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 &lt;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/)
description: 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 <br>   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<br>
   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