DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins

Microalgae are versatile organisms capable of converting CO2, H2O, and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity, and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity.

Selective disruption of an oncogenic mutant allele by CRISPR/Cas9 induces efficient tumor regression

Approximately 15% of non-small cell lung cancer cases are associated with a mutation in the epidermal growth factor receptor (EGFR) gene, which plays a critical role in tumor progression. With the goal of treating mutated EGFR-mediated lung cancer, we demonstrate the use of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system to discriminate between the oncogenic mutant and wild-type EGFR alleles and eliminate the carcinogenic mutant EGFR allele with high accuracy. We targeted an EGFR oncogene harboring a single-nucleotide missense mutation (CTG > CGG) that generates a protospacer-adjacent motif sequence recognized by the CRISPR/Cas9 derived from Streptococcus pyogenes. Co-delivery of Cas9 and an EGFR mutation-specific single-guide RNA via adenovirus resulted in precise disruption at the oncogenic mutation site with high specificity. Furthermore, this CRISPR/Cas9-mediated mutant allele disruption led to significantly enhanced cancer cell killing and reduced tumor size in a xenograft mouse model of human lung cancer. Taken together, these results indicate that targeting an oncogenic mutation using CRISPR/Cas9 offers a powerful surgical strategy to disrupt oncogenic mutations to treat cancers; similar strategies could be used to treat other mutation-associated diseases.

SIRT1-mediated downregulation of p27(Kip1) is essential for overcoming contact inhibition of Kaposi's sarcoma-associated herpesvirus transformed cells

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with Kaposi's sarcoma (KS), a malignancy commonly found in AIDS patients. Despite intensive studies in the last two decades, the mechanism of KSHV-induced cellular transformation and tumorigenesis remains unclear. In this study, we found that the expression of SIRT1, a metabolic sensor, was upregulated in a variety of KSHV-infected cells. In a model of KSHV-induced cellular transformation, SIRT1 knockdown with shRNAs or knockout by CRISPR/Cas9 gene editing dramatically suppressed cell proliferation and colony formation in soft agar of KSHV-transformed cells by inducing cell cycle arrest and contact inhibition. SIRT1 knockdown or knockout induced the expression of cyclin-dependent kinase inhibitor 1B (p27(Kip1)). Consequently, p27 knockdown rescued the inhibitory effect of SIRT1 knockdown or knockout on cell proliferation and colony formation. Furthermore, treatment of KSHV-transformed cells with a SIRT1 inhibitor, nicotinamide (NAM), had the same effect as SIRT1 knockdown and knockout. NAM significantly inhibited cell proliferation in culture and colony formation in soft agar, and induced cell cycle arrest. Significantly, NAM inhibited the progression of tumors and extended the survival of mice in a KSHV-induced tumor model. Collectively, these results demonstrate that SIRT1 suppression of p27 is required for KSHV-induced tumorigenesis and identify a potential therapeutic target for KS.

Direct observation of DNA target searching and cleavage by CRISPR-Cas12a

Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR-Cas9 for genome editing. Previous studies have revealed that Cas12a has unique features distinct from Cas9, but the detailed mechanisms of target searching and DNA cleavage by Cas12a are still unclear. Here, we directly observe this entire process by using single-molecule fluorescence assays to study Cas12a from Acidaminococcus sp. (AsCas12a). We determine that AsCas12a ribonucleoproteins search for their on-target site by a one-dimensional diffusion along elongated DNA molecules and induce cleavage in the two DNA strands in a well-defined order, beginning with the non-target strand. Furthermore, the protospacer-adjacent motif (PAM) for AsCas12a makes only a limited contribution of DNA unwinding during R-loop formation and shows a negligible role in the process of DNA cleavage, in contrast to the Cas9 PAM.

Web-based design and analysis tools for CRISPR base editing

Background: As a result of its simplicity and high efficiency, the CRISPR-Cas system has been widely used as a genome editing tool. Recently, CRISPR base editors, which consist of deactivated Cas9 (dCas9) or Cas9 nickase (nCas9) linked with a cytidine or a guanine deaminase, have been developed. Base editing tools will be very useful for gene correction because they can produce highly specific DNA substitutions without the introduction of any donor DNA, but dedicated web-based tools to facilitate the use of such tools have not yet been developed. Results: We present two web tools for base editors, named BE-Designer and BE-Analyzer. BE-Designer provides all possible base editor target sequences in a given input DNA sequence with useful information including potential off-target sites. BE-Analyzer, a tool for assessing base editing outcomes from next generation sequencing (NGS) data, provides information about mutations in a table and interactive graphs. Furthermore, because the tool runs client-side, large amounts of targeted deep sequencing data (< 1 GB) do not need to be uploaded to a server, substantially reducing running time and increasing data security. BE-Designer and BE-Analyzer can be freely accessed at http://www.rgenome.net/be-designer/ and http://www.rgenome.net/be-analyzer /, respectively. Conclusion: We develop two useful web tools to design target sequence (BE-Designer) and to analyze NGS data from experimental results (BE-Analyzer) for CRISPR base editors

Development of brain PET using GAPD arrays

Purpose: In recent times, there has been great interest in the use of Geiger-mode avalanche photodiodes (GAPDs) as scintillator readout in positron emission tomography (PET) detectors because of their advantages, such as high gain, compact size, low power consumption, and magnetic field insensitivity. The purpose of this study was to develop a novel PET system based on GAPD arrays for brain imaging. Methods: The PET consisted of 72 detector modules arranged in a ring of 330 mm diameter. Each PET module was composed of a 4 Â 4 matrix of 3 Â 3 Â 20 mm 3 cerium-doped lutetium yttrium orthosilicate (LYSO) crystals coupled with a 4 Â 4 array three-side tileable GAPD. The signals from each PET module were fed into preamplifiers using a 3 m long flat cable and then sent to a position decoder circuit (PDC), which output a digital address and an analog pulse of the interacted channel among 64 preamplifier signals tranmitted from four PET detector modules. The PDC outputs were fed into field programmable gate array (FPGA)-embedded data acquisition (DAQ) boards. The analog signal was then digitized, and arrival time and energy of the signal were calculated and stored. Results: The energy and coincidence timing resolutions measured for 511 keV gamma rays were 18.4 6 3.1% and 2.6 ns, respectively. The transaxial spatial resolution and sensitivity in the center of field of view (FOV) were 3.1 mm and 0.32% cps/Bq, respectively. The rods down to a diameter of 2.5 mm were resolved in a hot-rod phantom image, and activity distribution patterns between the white and gray matters in the Hoffman brain phantom were well imaged. Conclusions: Experimental results indicate that a PET system can be developed using GAPD arrays and the GAPD-based PET system can provide high-quality PET imaging

Reductive Collectivism and a Moral Justification for Killing in War

The purpose of this dissertation is to argue that most unjust combatants are complicitously liable to be killed while most unjust noncombatants are not liable to be killed, and to construct an adequate moral principle for satisfying these two desiderata based on reductive collectivism. The dissertation comprises four main chapters:Chapter Ⅰ is a preliminary discussion of this dissertation. I provide an overall understanding of Just War Theory and present Walzer’s key theses on killing in war. I then analyze the methodologies Walzer used to justify his claims. I also introduce the views of revisionists who challenge Walzer's view and explain in detail the methodological differences between reductive individualism and traditionalism. In Chapter Ⅱ, my main goal is to show that reductive individualism fails to satisfy two desiderata of an adequate moral principle regarding the justification of killing in war. In my view, if we assume that war is a conflict involving the use of armed force between collectives and these collectives are not adequately characterized solely in terms of relationships between individuals, then reductive individualism faces the individualized-liability dilemma. To that end, I explain McMahan’s individual liability-based account and demonstrate how the responsibility dilemma forces us to deny McMahan's account. I then argue that the prospects of reductive individualist accounts of liability solving the responsibility dilemma face serious problems. These problems give a good motivation for us to consider a collectivist account. In Chapter Ⅲ, I shift the main discussion from reductive individualism to reductive collectivism. Reductive collectivism makes use of a notion of collective action as opposed to individual action. I give a rough analysis of collective action and argue that what distinguishes genuine from non-genuine collective actions is a shared participatory intention among the members of the collective. I then clarify what it means to say that individuals who participate in collective action have a shared participatory intention. I also analyze how individual participatory intention is linked to the complicitous liability of individual agents in a collective. In Chapter Ⅳ, my primary goal is to evaluate the liability of both unjust combatants and unjust noncombatants in war. I argue that most unjust combatants are complicitously liable to be killed but most unjust noncombatants are not liable to be killed. In order to show that, the first part of this chapter is dedicated to constructing a moral principle, rooted in reductive collectivism, that determines who is complicitously liable for a collective unjust action. I then argue that members of an organization can have two different kinds of participatory intentions, and their complicitous liability is determined by what kind of intention an individual has as a collective member. In the second part of this chapter, I illustrate how the reductive collectivist moral principle that I draw satisfies the two desiderata for which I argue at the beginning of the chapter: that most unjust combatants are liable to be killed and that most unjust noncombatants are not liable to be killed