Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line - PubMed (original) (raw)
Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line
Patrick Essletzbichler et al. Genome Res. 2014 Dec.
Abstract
Near-haploid human cell lines are instrumental for genetic screens and genome engineering as gene inactivation is greatly facilitated by the absence of a second gene copy. However, no completely haploid human cell line has been described, hampering the genetic accessibility of a subset of genes. The near-haploid human cell line HAP1 contains a single copy of all chromosomes except for a heterozygous 30-megabase fragment of Chromosome 15. This large fragment encompasses 330 genes and is integrated on the long arm of Chromosome 19. Here, we employ a CRISPR/Cas9-based genome engineering strategy to excise this sizeable chromosomal fragment and to efficiently and reproducibly derive clones that retain their haploid state. Importantly, spectral karyotyping and single-nucleotide polymorphism (SNP) genotyping revealed that engineered-HAPloid (eHAP) cells are fully haploid with no gross chromosomal aberrations induced by Cas9. Furthermore, whole-genome sequence and transcriptome analysis of the parental HAP1 and an eHAP cell line showed that transcriptional changes are limited to the excised Chromosome 15 fragment. Together, we demonstrate the feasibility of efficiently engineering megabase deletions with the CRISPR/Cas9 technology and report the first fully haploid human cell line.
© 2014 Essletzbichler et al.; Published by Cold Spring Harbor Laboratory Press.
Figures
Figure 1.
Genomic makeup of HAP1 and strategy for deletion of the Chromosome 15 fragment. (A) HAP1 cells were subjected to spectral karyotyping to characterize their chromosomal landscape and identify disomic regions. (B) Single-nucleotide polymorphism data from KBM-7 cells were analyzed to identify chromosomal segments that are heterozygous and hence disomic. (Top) Heterozygous SNPs are depicted in red; homozygous SNPs, in turquoise. (Bottom) B-allele frequency (BAF) on Chromosome 15. A BAF of 0.5 is indicative of heterozygosity. (C) Schematic representative of the gRNA design. Two gRNAs (colored in pink) were placed at the boundaries of the disomic region from Chromosome 15.
Figure 2.
Combination of gRNAs 1 and 3 causes deletion of the Chromosome 15 fragment. HAP1 cells were transfected with various combinations of gRNAs (as indicated). Around 10 d post transfection, genomic DNA was isolated from pools of transfected cells. (A) The regions targeted by individual gRNAs were amplified by PCR using suitable primer pairs. Digestion of these PCR products by T7 endonuclease provides a semiquantitative measure for Cas9 editing efficiency. (B) To assess whether the fragment between gRNAs 1 and 3 had been excised following Cas9 cleavage, we performed a deletion PCR using a forward primer (HG6090) that binds to position Chr 15: 61,105,055 and a reverse primer (HG6093) that binds to position Chr 15: 89,889,818. We also included a control PCR (primer pair HG6090/HG6091) to confirm that every sample contained genomic DNA, suitable for PCR.
Figure 3.
Clones A11 and E9 are fully haploid human cell lines. (A) Deletion PCR (for experimental details, see Fig. 2B) for clones A11 and E9, as well as HAP1 wild-type cells. (B) To assess loss-of-heterozygosity in clones A11 and E9, we isolated genomic DNA and selected five genomic loci containing SNPs that were heterozygous in HAP1 cells. Each locus was amplified by suitable PCR primers and the PCR products were sent for Sanger sequencing. (C) Clones A11 and E9 were analyzed by spectral karyotyping to assess the global genomic landscape of these clones.
Figure 4.
Genomic and transcriptomic changes in eHAP cells are largely confined to Chromosome 15. (A) Whole-genome sequencing was performed on parental HAP1 cells and the A11 and E9 eHAP clones. In this panel, relative coverage between HAP1 and E9 data reveals a copy number loss restricted to the edited Chr 15 fragment. Large white regions correspond to unassembled pieces of the human genome. (B) Two biological replicates of HAP1 cells and two technical replicates of the E9 clone were subjected to RNA sequencing. Spearman’s correlation between the samples shows that overall expression is consistent between the parental line and the edited clones. (C) Two replicates of each cell line were compared pairwise. The number of highly expressed (FPKM > 5) and twofold differentially expressed genes are indicated. (D) Expression ratios between HAP1 and E9 cells were subjected to segmentation analysis. Red/blue bands show segments for which the expression level is consistently higher/lower in the parental cell line. (Inset) Details of the segmentation.
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