Quantitative subcellular reconstruction reveals a lipid mediated inter-organelle biogenesis network.

The structures and functions of organelles in cells depend on each other but have not been systematically explored. We established stable knockout cell lines of peroxisomal, Golgi and endoplasmic reticulum genes identified in a whole-genome CRISPR knockout screen for inducers of mitochondrial biogenesis stress, showing that defects in peroxisome, Golgi and endoplasmic reticulum metabolism disrupt mitochondrial structure and function. Our quantitative total-organelle profiling approach for focussed ion beam scanning electron microscopy revealed in unprecedented detail that specific organelle dysfunctions precipitate multi-organelle biogenesis defects, impair mitochondrial morphology and reduce respiration. Multi-omics profiling showed a unified proteome response and global shifts in lipid and glycoprotein homeostasis that are elicited when organelle biogenesis is compromised, and that the resulting mitochondrial dysfunction can be rescued with precursors for ether-glycerophospholipid metabolic pathways. This work defines metabolic and morphological interactions between organelles and how their perturbation can cause disease.

Copy number variation in tRNA isodecoder genes impairs mammalian development and balanced translation.

The number of tRNA isodecoders has increased dramatically in mammals, but the specific molecular and physiological reasons for this expansion remain elusive. To address this fundamental question we used CRISPR editing to knockout the seven-membered phenylalanine tRNA gene family in mice, both individually and combinatorially. Using ATAC-Seq, RNA-seq, ribo-profiling and proteomics we observed distinct molecular consequences of single tRNA deletions. We show that tRNA-Phe-1-1 is required for neuronal function and its loss is partially compensated by increased expression of other tRNAs but results in mistranslation. In contrast, the other tRNA-Phe isodecoder genes buffer the loss of each of the remaining six tRNA-Phe genes. In the tRNA-Phe gene family, the expression of at least six tRNA-Phe alleles is required for embryonic viability and tRNA-Phe-1-1 is most important for development and survival. Our results reveal that the multi-copy configuration of tRNA genes is required to buffer translation and viability in mammals.

Multi-omic profiling reveals an RNA processing rheostat that predisposes to prostate cancer.

Prostate cancer is the most commonly diagnosed malignancy and the third leading cause of cancer deaths. GWAS have identified variants associated with prostate cancer susceptibility; however, mechanistic and functional validation of these mutations is lacking. We used CRISPR-Cas9 genome editing to introduce a missense variant identified in the ELAC2 gene, which encodes a dually localised nuclear and mitochondrial RNA processing enzyme, into the mouse Elac2 gene as well as to generate a prostate-specific knockout of Elac2. These mutations caused enlargement and inflammation of the prostate and nodule formation. The Elac2 variant or knockout mice on the background of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model show that Elac2 mutation with a secondary genetic insult exacerbated the onset and progression of prostate cancer. Multiomic profiling revealed defects in energy metabolism that activated proinflammatory and tumorigenic pathways as a consequence of impaired noncoding RNA processing and reduced protein synthesis. Our physiologically relevant models show that the ELAC2 variant is a predisposing factor for prostate cancer and identify changes that underlie the pathogenesis of this cancer.

Computationally designed hyperactive Cas9 enzymes.

The ability to alter the genomes of living cells is key to understanding how genes influence the functions of organisms and will be critical to modify living systems for useful purposes. However, this promise has long been limited by the technical challenges involved in genetic engineering. Recent advances in gene editing have bypassed some of these challenges but they are still far from ideal. Here we use FuncLib to computationally design Cas9 enzymes with substantially higher donor-independent editing activities. We use genetic circuits linked to cell survival in yeast to quantify Cas9 activity and discover synergistic interactions between engineered regions. These hyperactive Cas9 variants function efficiently in mammalian cells and introduce larger and more diverse pools of insertions and deletions into targeted genomic regions, providing tools to enhance and expand the possible applications of CRISPR-based gene editing.

In silico evolution of nucleic acid-binding proteins from a nonfunctional scaffold.

Directed evolution emulates the process of natural selection to produce proteins with improved or altered functions. These approaches have proven to be very powerful but are technically challenging and particularly time and resource intensive. To bypass these limitations, we constructed a system to perform the entire process of directed evolution in silico. We employed iterative computational cycles of mutation and evaluation to predict mutations that confer high-affinity binding activities for DNA and RNA to an initial de novo designed protein with no inherent function. Beneficial mutations revealed modes of nucleic acid recognition not previously observed in natural proteins, highlighting the ability of computational directed evolution to access new molecular functions. Furthermore, the process by which new functions were obtained closely resembles natural evolution and can provide insights into the contributions of mutation rate, population size and selective pressure on functionalization of macromolecules in nature.