Identification and characterization of essential genes in the human genome

Abstract

Large-scale genetic analysis of lethal phenotypes has elucidated the molecular underpinnings of many biological processes. Using the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) system, we constructed a genome-wide single-guide RNA library to screen for genes required for proliferation and survival in a human cancer cell line. Our screen revealed the set of cell-essential genes, which was validated with an orthogonal gene-trap-based screen and comparison with yeast gene knockouts. This set is enriched for genes that encode components of fundamental pathways, are expressed at high levels, and contain few inactivating polymorphisms in the human population. We also uncovered a large group of uncharacterized genes involved in RNA processing, a number of whose products localize to the nucleolus. Last, screens in additional cell lines showed a high degree of overlap in gene essentiality but also revealed differences specific to each cell line and cancer type that reflect the developmental origin, oncogenic drivers, paralogous gene expression pattern, and chromosomal structure of each line. These results demonstrate the power of CRISPR-based screens and suggest a general strategy for identifying liabilities in cancer cells.

Fig. 1. Two approaches for genetic screening in human cells

CRISPR/Cas9 (top): Cells are transduced with a genome-wide sgRNA lentiviral library. Gene inactivation via Cas9-mediated genomic cleavage is directed by the 20-bp sequence at the 5′ end of the sgRNA. Cells bearing sgRNAs targeting essential genes are depleted in the final population. Gene-trap (bottom): KBM7 cells are transduced with a gene-trap retrovirus which integrates in an inactivating or “harmless” orientation at random genomic loci. Essential genes contain fewer insertions in the inactivating orientation. Sample data for two neighboring genes RPL14, encoding an essential ribosomal protein, and ZNF619, encoding a dispensable zinc finger protein, are displayed. For CRISPR/Cas9, sgRNAs are plotted according to their target position along each gene with the height of each bar indicating the level of depletion. Boxes indicate individual exons. For gene-trap, the intronic insertion sites in each gene are plotted according to their orientation and genomic position. The height of each point is randomized.

Fig. 2. Identification and characterization of human cell-essential genes

(A) CRISPR scores (CS) of all genes in the KBM7 cells. Similar proportions of cell-essential genes were identified on all autosomes. (B) KBM7 gene-trap score (GTS) distributions. No low GTS genes were detected on the diploid chromosome 8. (C) CS and GTS of overlapping genes. (D) Yeast homolog essentiality prediction analysis. (E) Broader retention of essential genes across species. (F) Higher sequence conservation of essential genes. (G) Genes with deleterious stop-gain variants are less likely to be essential. (H) Greater connectivity of proteins encoded by essential genes. (I) Higher mRNA transcript levels of essential genes. (J) Genes with paralogs are less likely to be essential. *** denotes p<0.001 from Kolmogorov-Smirnov test.

Fig. 3. Functional characterization of novel cell-essential genes

(A) Of the 1,878 cell-essential genes identified in the KBM7 cell line, 330 genes had no known function. (B) Genes co-expressed with C16orf80, C3orf17, and C9orf114 across CCLE cell lines were associated with RNA processing. Parentheses denote the number of genes in the set. (C) Proliferation of KBM7 cells transduced with sgRNAs targeting C16orf80, C3orf17, and C9orf114, or a non-targeting control. Error bars denote SD (n=4). (D) C16orf80 localized to the nucleus and C3orf17 and C9orf114 to the nucleolus. Fibrillarin-RFP was used as a nucleolar marker. (E) Multiple subunits of the spliceosome, RNAse P/MRP and H/ACA ribonucleoprotein complexes interact with C16orf80, C3orf17, and C9orf114, respectively.

Fig. 4. Comparisons of gene essentiality across four cell lines

(A) Heatmap of CS across four cell lines sorted by average CS. (B) CS of genes residing in the non-pseudoautosomal region of chromosome Y for male cell lines (Raji, Jiyoye and KBM7). (C) Sanger sequencing of DDX3X in Raji cells reveals mutations in the 5′ splice site of intron 8. (D) Splice-site mutations in DDX3X result in a 69-bp truncation of the mRNA. PCR primers spanning exons 8 and 9 of DDX3X (denoted by green arrows) were used to amplify cDNA from each line. (E) Essentiality of DDX3X and DDX3Y is determined by the expression and mutation status of their paralogs. (F) Proliferation of GFP- and DDX3X-expressing Raji cells transduced with sgRNAs targeting DDX3Y or an AAVS1-targeting control. Error bars denote SD (n=4). (G) Analysis of genes on chromosome 22q reveals apparent ‘essentiality’ of 61 contiguous genes in K562 residing in a region of high-copy tandem amplification. (H) Proliferation of K562 and KBM7 cells transduced with sgRNAs targeting a non-genic region within the BCR-ABL amplicon in K562 cells or a non-targeting control. Error bars denote SD (n=4). (I) γH2AX (phospho-S139 H2AX) immunoblot analysis of K562 transduced with sgRNAs as in (H). S6K1 was used as a loading control. (J) Cleavage within amplified region induced erythroid differentiation in K562 cells as assessed by 3,3′-dimethoxybenzidine hemoglobin staining. (K) Comparison of gene essentiality between the two cancer types reveals oncogenic drivers and lineage specifiers. Genes were ranked by the difference between the average CS of each cancer type.