Highly parallel genome variant engineering with CRISPR-Cas9.

Understanding the functional effects of DNA sequence variants is of critical importance for studies of basic biology, evolution, and medical genetics; however, measuring these effects in a high-throughput manner is a major challenge. One promising avenue is precise editing with the CRISPR-Cas9 system, which allows for generation of DNA double-strand breaks (DSBs) at genomic sites matching the targeting sequence of a guide RNA (gRNA). Recent studies have used CRISPR libraries to generate many frameshift mutations genome wide through faulty repair of CRISPR-directed breaks by nonhomologous end joining (NHEJ) 1 . Here, we developed a CRISPR-library-based approach for highly efficient and precise genome-wide variant engineering. We used our method to examine the functional consequences of premature-termination codons (PTCs) at different locations within all annotated essential genes in yeast. We found that most PTCs were highly deleterious unless they occurred close to the 3' end of the gene and did not affect an annotated protein domain. Unexpectedly, we discovered that some putatively essential genes are dispensable, whereas others have large dispensable regions. This approach can be used to profile the effects of large classes of variants in a high-throughput manner.

New insights into the troubles of aneuploidy.

Deviation from a balanced genome by either gain or loss of entire chromosomes is generally tolerated poorly in all eukaryotic systems studied to date. Errors in mitotic or meiotic cell division lead to aneuploidy, which places a burden of additional or insufficient gene products from the missegregated chromosomes on the daughter cells. The burden of aneuploidy often manifests itself as impaired fitness of individual cells and whole organisms, in which abnormal development is also characteristic. However, most human cancers, noted for their rapid growth, also display various levels of aneuploidy. Here we discuss the detrimental, potentially beneficial, and sometimes puzzling effects of aneuploidy on cellular and organismal fitness and tissue function as well as its role in diseases such as cancer and neurodegeneration.

Identification of aneuploidy-selective antiproliferation compounds.

Aneuploidy, an incorrect chromosome number, is a hallmark of cancer. Compounds that cause lethality in aneuploid, but not euploid, cells could therefore provide new cancer therapies. We have identified the energy stress-inducing agent AICAR, the protein folding inhibitor 17-AAG, and the autophagy inhibitor chloroquine as exhibiting this property. AICAR induces p53-mediated apoptosis in primary mouse embryonic fibroblasts (MEFs) trisomic for chromosome 1, 13, 16, or 19. AICAR and 17-AAG, especially when combined, also show efficacy against aneuploid human cancer cell lines. Our results suggest that compounds that interfere with pathways that are essential for the survival of aneuploid cells could serve as a new treatment strategy against a broad spectrum of human tumors.

Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking.

The nuclear pore complex (NPC) mediates all exchange between the cytoplasm and the nucleus. Small molecules can passively diffuse through the NPC, whereas larger cargos require transport receptors to translocate. How the NPC facilitates the translocation of transport receptor/cargo complexes remains unclear. To investigate this process, we tracked single protein-functionalized quantum dot cargos as they moved through human NPCs. Here we show that import proceeds by successive substeps comprising cargo capture, filtering and translocation, and release into the nucleus. Most quantum dots are rejected at one of these steps and return to the cytoplasm, including very large cargos that abort at a size-selective barrier. Cargo movement in the central channel is subdiffusive and cargos that can bind more transport receptors diffuse more freely. Without Ran GTPase, a critical regulator of transport directionality, cargos still explore the entire NPC, but have a markedly reduced probability of exit into the nucleus, suggesting that NPC entry and exit steps are not equivalent and that the pore is functionally asymmetric to importing cargos. The overall selectivity of the NPC seems to arise from the cumulative action of multiple reversible substeps and a final irreversible exit step.

Optical measurement of mechanical forces inside short DNA loops.

Knowledge of the mechanical properties of double-stranded DNA (dsDNA) is essential to understand the role of dsDNA looping in gene regulation and the mechanochemistry of molecular machines that operate on dsDNA. Here, we use a newly developed tool, force sensors with optical readout, to measure the forces inside short, strained loops composed of both dsDNA and single-stranded DNA. By varying the length of the loops and their proportion of dsDNA, it was possible to vary their internal forces from 1 pN to >20 pN. Surprisingly, internal loop forces changed erratically as the amount of dsDNA was increased for a given loop length, with the effect most notable in the smallest loop (57 nucleotides). Monte Carlo simulations based on the helical wormlike chain model accurately predict internal forces when more than half of the loop is dsDNA but fail otherwise. Mismatches engineered into the double-stranded regions increased flexibility, suggesting that Watson-Crick basepaired dsDNA can withstand high compressive forces without recourse to multibase melts. Fluorescence correlation spectroscopy further excluded transient melting (microsecond to millisecond duration) as a mechanism for relief of compressive forces in the tested dsDNAs. DNA loops with integrated force sensors may allow the comprehensive mapping of the elasticity of short dsDNAs as a function of both sequence and salt.