CRISPR-Cas genome engineering in yeast has relied on preparation of complex expression plasmids for multiplexed gene knockouts and point mutations. Here we show that co-transformation of a single linearized plasmid with multiple PCR-generated guide RNA (gRNA) and donor DNA cassettes facilitates high-efficiency multiplexed integration of point mutations and large constructs. This technique allowed recovery of marker-less triple-engineering events with 64% efficiency without selection for expression of all gRNAs. The gRNA cassettes can be easily made by PCR and delivered in any combination. We employed this method to rapidly phenotype up to five specific allele combinations and identify synergistic effects. To prototype a pathway for the production of muconic acid, we integrated six DNA fragments totaling 24 kb across three loci in naive Saccharomyces cerevisiae in a single transformation. With minor modifications, we integrated a similar pathway in Kluyveromyces lactis. The flexibility afforded by combinatorial gRNA delivery dramatically accelerates complex strain engineering for basic research and industrial fermentation.

The living cell is an incredibly complex entity, and the goal of predictively and quantitatively understanding its function is one of the next great challenges in biology. Much of what we know about the cell concerns its constituent parts, but to a great extent, we have yet to decode how these parts are organized to yield complex physiological function. Classically, we have learned about the organization of cellular networks by perturbing them through genetic or chemical means. The emerging discipline of synthetic biology offers an additional, powerful way to study systems. By rearranging the parts that comprise existing networks, we can gain valuable insight into the hierarchical logic of the networks and identify the modular building blocks that evolution uses to generate innovative function. Additionally, by building minimal "toy" networks, one can systematically explore the relationship between network space (linkages and parameters) and functional space (the system's physiological behavior). Here, we outline recent work that uses synthetic biology approaches to investigate the organization and function of cellular networks, and describe a vision for a synthetic biology toolkit that could be used to interrogate the design principles of diverse systems.

The breast-and ovarian-specific tumor suppressor BRCA1, when associated with BARD1, is an ubiquitin ligase. We have shown here that this heterodimer ubiquitinates a hyperphosphorylated form of Rpb1, the largest subunit of RNA polymerase II. Two major phosphorylation sites have been identified in the Rpb1 carboxyl terminal domain, serine 2 (Ser-2) or serine 5 (Ser-5) of the YSPTSPS heptapeptide repeat. Only the Ser-5 hyperphosphorylated form is ubiquitinated by BRCA1/ BARD1. Overexpression of BRCA1 in cells stimulated the DNA damage-induced ubiquitination of Rpb1. Similar to the in vitro reaction, the stimulation of Rpb1 ubiquitination by BRCA1 in cells occurred only on those molecules hyperphosphorylated on Ser-5 of the heptapeptide repeat. In vitro, the carboxyl terminus of BRCA1 (amino acids 501-1863) was dispensable for the ubiquitination of hyperphosphorylated Rpb1. In cells, however, efficient Rpb1 ubiquitination required the carboxyl terminus of BRCA1, suggesting that interactions mediated by this region were essential in the complex milieu of the nucleus. These results link the BRCA1-dependent ubiquitination of the polymerase with DNA damage.BRCA1, the breast-and ovarian-specific tumor suppressor protein, has been found to regulate a number of processes central to the normal function of the cell, including transcription, chromatin dynamics, homologous recombination, and other forms of DNA damage repair (1, 2). Because BRCA1 has been found associated with a wide range of proteins involved in these processes, it may function as a scaffold, organizing effector proteins in a context-dependent manner. However, when BRCA1 is associated with the BARD1 protein, it is also an enzyme, an E3 ubiquitin ligase (3, 4). The realization that BRCA1 is an enzyme establishes the necessity of identifying its substrates in order to understand how the ubiquitination activity impacts these processes in the cell.BRCA1 and BARD1 are associated with the messenger RNAsynthesizing polymerase in a complex known as the RNA polymerase II holoenzyme (holo-pol) 1 (5-7). One function for BRCA1 in this holo-pol complex appears to be as a coactivator of transcription, because it has been shown that BRCA1 stimulates the activation signal of p53, NF-B, and others (8 -13). Previously, we modeled that the BRCA1 and BARD1 in the holo-pol complex may ubiquitinate the transcribing RNA polymerase II (RNAPII) when it encounters DNA damage, and we also suggested that this ubiquitination event would stimulate the repair process (14,15).Rpb1 is the largest subunit of RNAPII, and its carboxylterminal domain (CTD) is highly conserved, consisting of multiple repeats (27 in budding yeast, 52 in humans) of the heptapeptide YSPTSPS. Serines 2 (Ser-2) and 5 (Ser-5) of multiple repeats are phosphorylated co-transcriptionally, Ser5*p predominating at the promoter and Ser2*p in the coding sequence (16,17). In response to DNA damage Rpb1 is also ubiquitinated, an event associated with changes in concentration of both the hypophosphorylated and the hyperphosphoryl...

Prior studies provide data supporting the notion that ATP binding cassette transporter A1 (ABCA1) promotes lipid efflux to extracellular acceptors in a two-step process: first, ABCA1 mediates phospholipid efflux to an apolipoprotein, and second, this apolipoprotein-phospholipid complex accepts free cholesterol in an ABCA1-independent manner. In the current study using RAW264.7 cells, ABCA1-mediated free cholesterol and phospholipid efflux to apolipoprotein A-I (apoA-I) were tightly coupled to each other both temporally and after treatment with ABCA1 inhibitors. The time course and temperature dependence of ABCA1-mediated lipid efflux to apoA-I support a role for endocytosis in this process. Cyclodextrin treatment of RAW264.7 cells partially inhibited 8Br-cAMP-induced efflux of free cholesterol and phospholipid to apoA-I. ABCA1-expressing cells are more sensitive to cell damage by highdose cyclodextrin and vanadate, leading to increased lactate dehydrogenase leakage and phospholipid release even in the absence of the acceptor apoA-I. Finally, we could not reproduce a two-step effect on lipid efflux using conditioned medium from ABCA1-expressing cells pretreated with cyclodextrin. Cellular expression of ATP binding cassette transporter A1 (ABCA1) promotes the efflux of both free cholesterol (FC) and phospholipids (PLs) to extracellular acceptors such as apolipoprotein A-I (apoA-I); however, the mechanism of this efflux is not understood. Fielding et al. (1) proposed a two-step mechanism in which ABCA1 mediates PL efflux to apoA-I, which in turn can then pick up FC in an ABCA1-independent autocrine or paracrine manner. This conclusion was based on two types of experiments: 1 ) PL efflux from vascular smooth muscle cells to apoA-I is less sensitive to vanadate inhibition than FC efflux, and 2 ) medium containing apoA-I that is conditioned on smooth muscle cells can lead to FC efflux from vascular endothelial cells that do not express ABCA1 (1). Wang et al. (2) provided evidence for the two-step pathway by demonstrating that 1 ) a 30 min pretreatment of ABCA1-expressing cells with 20 mM 2-hydroxypropyl-␤ -cyclodextrin reduced FC efflux to apoA-I without reducing PL efflux, and 2 ) medium containing apoA-I that is conditioned on cyclodextrin-pretreated ABCA1-expressing cells could lead to FC efflux from cells that do not express ABCA1. These experiments and others from Chimini and colleagues (3, 4), who demonstrated that ABCA1 could mediate phosphatidylserine translocase activity, have led to the notion that the primary activity of ABCA1 is the assembly of PL onto acceptors and that FC efflux follows passively by a mechanism not dependent on ABCA1.We demonstrate here that FC and PL efflux to apoA-I is concurrent in the RAW264.7 murine macrophage cell line, in which ABCA1 expression is inducible by cAMP analogs (5, 6). ABCA1-mediated lipid efflux has delayed kinetics and is abolished at room temperature, results that are consistent with the need for endocytosis and vesicular trafficking for efflux to occur. We...

The following two theories for the mechanism of ABCA1 in lipid efflux to apolipoprotein acceptors have been proposed: 1) that ABCA1 directly binds the apolipoprotein ligand and then facilitates lipid efflux and 2) that ABCA1 acts as a phosphatidylserine (PS) translocase, increasing PS levels in the plasma membrane exofacial leaflet, and that this is sufficient to facilitate apolipoprotein binding and lipid assembly. Upon induction of ABCA1 in RAW264.7 cells by cAMP analogues there was a moderate increase in cell surface PS as detected by annexin V binding, whereas apoAI binding was increased more robustly. Apoptosis induced large increases in annexin V and apoAI binding; however, apoptotic cells did not efflux lipids to apoAI. Annexin V did not act as a cholesterol acceptor, and it did not compete for the cholesterol acceptor or cell binding activity of apoAI. ApoAI binds to ABCA1-expressing cells, and with incubation at 37°C apoAI is co-localized within the cells in ABCA1-containing endosomes. Fluorescent recovery after photobleaching demonstrated that apoAI bound to ABCA1-expressing cells was relatively immobile, suggesting that it was bound either directly or indirectly to an integral membrane protein. Although ABCA1 induction was associated with a small increase in cell surface PS, these results argue against the notion that this cell surface PS is sufficient to mediate cellular apoAI binding and lipid efflux.The ATP binding cassette (ABC) 1 family of proteins serves to pump a diverse set of molecules out of cells. ABCA1, the Tangier disease gene, is required for cholesterol and phospholipid efflux to lipid-free apolipoproteins (for review see Ref. 1). The exact mechanism of this lipid efflux pump and the location of the lipid transfer and assembly onto apolipoprotein acceptors have yet to be determined definitively. It has been shown previously that ABCA1 expression leads to increased cell surface binding and uptake of apoAI (2). Protein cross-linking studies have shown that cell surface ABCA1 is closely associated with exogenously added apoAI (3, 4). Thus, one theory of ABCA1 action is that the apolipoprotein ligand is bound directly to the ABCA1 receptor, which can then mediate lipid assembly to form a nascent lipoprotein. An alternative theory of ABCA1 function has been put forth by Chimini and coworkers (5, 6). Phosphatidylserine (PS) is normally asymmetrically distributed on the plasma membrane such that most of it is sequestered on the inner leaflet. Increased levels of PS in the exofacial leaflet have been demonstrated in ABCA1 transiently transfected cells, thus providing evidence that ABCA1 may function as a PS translocase to pump PS from the cytoplasmic leaflet to the exofacial leaflet (5).This activity is referred to as "floppase" activity to distinguish it from the "flippase" activity that pumps PS from the outer to inner plasma membrane leaflets (7). These authors propose that PS translocase activity is sufficient to lead to apoAI binding to and lipid efflux from ABCA1-expressing cells and that...