Publications by authors named "Zohra Kalani"

4 Publications

  • Page 1 of 1

Noncanonical open reading frames encode functional proteins essential for cancer cell survival.

Nat Biotechnol 2021 Jan 28. Epub 2021 Jan 28.

Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Although genomic analyses predict many noncanonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here we experimentally interrogated 553 candidates selected from noncanonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Following ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. Clustered regularly interspaced short palindromic repeat (CRISPR) tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442-renamed glycine-rich extracellular protein-1 (GREP1)-encodes a secreted protein highly expressed in breast cancer, and its knockout in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. Our experiments suggest that noncanonical ORFs can express biologically active proteins that are potential therapeutic targets.
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http://dx.doi.org/10.1038/s41587-020-00806-2DOI Listing
January 2021

A Compendium of Genetic Modifiers of Mitochondrial Dysfunction Reveals Intra-organelle Buffering.

Cell 2019 11;179(5):1222-1238.e17

Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114, USA, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA. Electronic address:

Mitochondrial dysfunction is associated with a spectrum of human conditions, ranging from rare, inborn errors of metabolism to the aging process. To identify pathways that modify mitochondrial dysfunction, we performed genome-wide CRISPR screens in the presence of small-molecule mitochondrial inhibitors. We report a compendium of chemical-genetic interactions involving 191 distinct genetic modifiers, including 38 that are synthetic sick/lethal and 63 that are suppressors. Genes involved in glycolysis (PFKP), pentose phosphate pathway (G6PD), and defense against lipid peroxidation (GPX4) scored high as synthetic sick/lethal. A surprisingly large fraction of suppressors are pathway intrinsic and encode mitochondrial proteins. A striking example of such "intra-organelle" buffering is the alleviation of a chemical defect in complex V by simultaneous inhibition of complex I, which benefits cells by rebalancing redox cofactors, increasing reductive carboxylation, and promoting glycolysis. Perhaps paradoxically, certain forms of mitochondrial dysfunction may best be buffered with "second site" inhibitors to the organelle.
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http://dx.doi.org/10.1016/j.cell.2019.10.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053407PMC
November 2019

Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens.

Nat Biotechnol 2018 02 18;36(2):179-189. Epub 2017 Dec 18.

Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.

Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The "Big Papi" (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.
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http://dx.doi.org/10.1038/nbt.4048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800952PMC
February 2018

Computational correction of copy number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells.

Nat Genet 2017 Dec 30;49(12):1779-1784. Epub 2017 Oct 30.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

The CRISPR-Cas9 system has revolutionized gene editing both at single genes and in multiplexed loss-of-function screens, thus enabling precise genome-scale identification of genes essential for proliferation and survival of cancer cells. However, previous studies have reported that a gene-independent antiproliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, thereby leading to false-positive results in copy number-amplified regions. We developed CERES, a computational method to estimate gene-dependency levels from CRISPR-Cas9 essentiality screens while accounting for the copy number-specific effect. In our efforts to define a cancer dependency map, we performed genome-scale CRISPR-Cas9 essentiality screens across 342 cancer cell lines and applied CERES to this data set. We found that CERES decreased false-positive results and estimated sgRNA activity for both this data set and previously published screens performed with different sgRNA libraries. We further demonstrate the utility of this collection of screens, after CERES correction, for identifying cancer-type-specific vulnerabilities.
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http://dx.doi.org/10.1038/ng.3984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709193PMC
December 2017