Engineering an anaerobic derivative of the obligately aerobic thermophile Thermus thermophilus HB27 for the purposes of studying thermophilic aerobic respiratory chain proteins

The thermophilic bacterium Thermus thermophilus (Tth) is an obligate aerobe with many important biotechnological applications and products. Thermostable proteins isolated from Thermus species have proven important in understanding adaptation to thermophilic environments. Thermophilic adaptations of respiratory electron transport chains (ETC) are not well characterized. Conventional homology-based, genetic, and biochemical techniques are limited in their ability to identify thermophilic Tth homologues of mesophilic respiratory ETC proteins due to the lack of a mutable facultative platform. We are attempting to generate this platform by increasing glycolytic flux through metabolic engineering techniques. Based on physiological studies, Tth HB27 demonstrates a low affinity for monosaccharide substrates, which limits glycolytic flux. We identify growth conditions suitable to increasing metabolic flux, as well as limitations to our current method for engineering a Tth mutable facultative platform. We also propose future direction in order to generate anaerobic derivatives of Tth HB27.

Aag DNA Glycosylase Promotes Alkylation-Induced Tissue Damage Mediated by Parp1

Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag−/− mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.National Institutes of Health (U.S.) (NIH grant R01-CA075576)National Institutes of Health (U.S.) (NIH grant R01-CA055042)National Institutes of Health (U.S.) (NIH grant R01-CA149261)National Institutes of Health (U.S.) (NIH grant P30-ES00002)National Institutes of Health (U.S.) (NIH grant P30-ES02109)National Center for Research Resources (U.S.) (grant number M01RR-01066)National Center for Research Resources (U.S.) (grant number UL1 RR025758, Harvard Clinical and Translational Science Center

Reactivating Latent HIV-1

Complete eradication of HIV-1 infection is impeded by the existence of cells that harbor chromosomally integrated but transcriptionally inactive provirus. These latently-infected cells can persist for years without producing viral progeny, rendering them refractory to immune surveillance and antiretroviral therapy and providing a permanent reservoir for the stochastic reactivation and reseeding of HIV-1. Strategies for purging this latent reservoir are thus needed to eradicate infection. Here we show that engineered transcriptional activation systems based on CRISPR/Cas9 can be harnessed to activate HIV-1 expression in cell line models of latency. We utilized two distinct CRISPR transcriptional activation systems, dCas9-VP64 and the synergistic activation mediator (SAM) complex, to target numerous sites across the HIV-1 long terminal repeat (LTR) promoter and observed robust expression from the full-length HIV-1 promoter in multiple cell line models of HIV-1 latency. We further demonstrated that complementing Cas9 activators with latency-reversing compounds can enhance latent HIV-1 transcription and that epigenome modulation using CRISPR-based acetyltransferases could also promote viral gene activation. Finally, we showed that latent HIV-1 expression could also be stimulated by CRISPR-mediated activation of endogenous factors not previously implicated in HIV-1 pathogenesis but whose expression could nonetheless reactivate viral gene expression. Collectively, these results demonstrate that CRISPR systems are potentially effective tools for inducing latent HIV-1 expression and that their use, in combination with antiretroviral therapy, could lead to improved therapies for HIV-1 infection

Genome-wide activation screens to increase adeno-associated virus production.

We describe a genome-wide screening strategy to identify target genes whose modulation increases the capacity of a cell to produce recombinant adeno-associated viral (AAV) vector. Specifically, a single-guide RNA (sgRNA) library for a CRISPR-based genome-wide transcriptional activation screen was inserted into an AAV vector, and iterative rounds of viral infection and rescue in HEK293 producer cells enabled the enrichment of sgRNAs targeting genes whose upregulation increased AAV production. Numerous gain-of-function targets were identified, including spindle and kinetochore associated complex subunit 2 (SKA2) and inositol 1, 4, 5-trisphosphate receptor interacting protein (ITPRIP). Furthermore, individual or combinatorial modulation of these targets in stable producer cell lines increased vector genomic replication and loading into AAV virions, resulting in up to a 3.8-fold increase in AAV manufacturing capacity. Our study offers an efficient approach to engineer viral vector producer cell lines and enhances our understanding of the roles of SKA2 and ITPRIP in AAV packaging

In vivo genome editing improves motor function and extends survival in a mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease characterized by the progressive loss of motor neurons in the spinal cord and brain. In particular, autosomal dominant mutations in the superoxide dismutase 1 (SOD1) gene are responsible for ~20% of all familial ALS cases. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas9) genome editing system holds the potential to treat autosomal dominant disorders by facilitating the introduction of frameshift-induced mutations that can disable mutant gene function. We demonstrate that CRISPR-Cas9 can be harnessed to disrupt mutant SOD1 expression in the G93A-SOD1 mouse model of ALS following in vivo delivery using an adeno-associated virus vector. Genome editing reduced mutant SOD1 protein by >2.5-fold in the lumbar and thoracic spinal cord, resulting in improved motor function and reduced muscle atrophy. Crucially, ALS mice treated by CRISPR-mediated genome editing had ~50% more motor neurons at end stage and displayed a ~37% delay in disease onset and a ~25% increase in survival compared to control animals. Thus, this study illustrates the potential for CRISPR-Cas9 to treat SOD1-linked forms of ALS and other central nervous system disorders caused by autosomal dominant mutations

Orthogonal control of expression mean and variance by epigenetic features at different genomic loci.

While gene expression noise has been shown to drive dramatic phenotypic variations, the molecular basis for this variability in mammalian systems is not well understood. Gene expression has been shown to be regulated by promoter architecture and the associated chromatin environment. However, the exact contribution of these two factors in regulating expression noise has not been explored. Using a dual-reporter lentiviral model system, we deconvolved the influence of the promoter sequence to systematically study the contribution of the chromatin environment at different genomic locations in regulating expression noise. By integrating a large-scale analysis to quantify mRNA levels by smFISH and protein levels by flow cytometry in single cells, we found that mean expression and noise are uncorrelated across genomic locations. Furthermore, we showed that this independence could be explained by the orthogonal control of mean expression by the transcript burst size and noise by the burst frequency. Finally, we showed that genomic locations displaying higher expression noise are associated with more repressed chromatin, thereby indicating the contribution of the chromatin environment in regulating expression noise

Characterization of γ-Butyrolactone Autoregulatory Signaling Gene Homologs in the Angucyclinone Polyketide WS5995B Producer Streptomyces acidiscabies▿

Organisms belonging to the genus Streptomyces produce numerous important secondary metabolites and undergo a sophisticated morphological differentiation program. In many instances these processes are under the control of γ-butyrolactone (GBL) autoregulatory systems. Streptomyces acidiscabies strain 84.104 produces the secondary metabolite aromatic angucyclinone polyketide WS5995B. In order to explore the role of GBL regulatory circuitry in WS5995B production and morphogenesis in S. acidiscabies, a gene cluster encoding GBL autoregulatory signaling homologs was identified and characterized. Two GBL receptor homologs, sabR and sabS, were found flanking a GBL synthase homolog sabA. Strains carrying mutations in sabS produced elevated levels of WS5995B and displayed conditional morphological defects reminiscent of defects seen in Streptomyces bldA mutants. Notably, sabS possesses a TTA codon predicted to be recognized by tRNAleu. sabA mutants produced higher levels of WS5995B than the wild-type strain but to a lesser extent than the levels of WS5995B seen in sabS mutants. Purified recombinant SabR and SabS were tested for their abilities to bind predicted AT-rich autoregulatory element (ARE) boxes within the sabRAS region. SabS did not bind any DNA sequences in this region, while SabR bound an ARE box in the region upstream of sabS. Quantitative reverse transcription-PCR analysis revealed higher levels of sabS transcript in sabR mutants than in the wild-type strain, suggesting that sabS expression is repressed by SabR. Based on these data, we propose that the S. acidiscabies sabRAS genes encode components of a signaling pathway which participates in the regulation of WS5995B production and morphogenesis

Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery.

Realizing the full potential of genome editing requires the development of efficient and broadly applicable methods for delivering programmable nucleases and donor templates for homology-directed repair (HDR). The RNA-guided Cas9 endonuclease can be introduced into cells as a purified protein in complex with a single guide RNA (sgRNA). Such ribonucleoproteins (RNPs) can facilitate the high-fidelity introduction of single-base substitutions via HDR following co-delivery with a single-stranded DNA oligonucleotide. However, combining RNPs with transgene-containing donor templates for targeted gene addition has proven challenging, which in turn has limited the capabilities of the RNP-mediated genome editing toolbox. Here, we demonstrate that combining RNP delivery with naturally recombinogenic adeno-associated virus (AAV) donor vectors enables site-specific gene insertion by homology-directed genome editing. Compared to conventional plasmid-based expression vectors and donor templates, we show that combining RNP and AAV donor delivery increases the efficiency of gene addition by up to 12-fold, enabling the creation of lineage reporters that can be used to track the conversion of striatal neurons from human fibroblasts in real time. These results thus illustrate the potential for unifying nuclease protein delivery with AAV donor vectors for homology-directed genome editing