Cas9-based Genome-editing Tools Research Articles

Genome Editing of the Anaerobic Thermophile Thermoanaerobacter ethanolicus Using Thermostable Cas9.

Thermoanaerobacter ethanolicus can produce acetate, lactate, hydrogen, and ethanol from sugars resulting from plant carbohydrate polymer degradation at temperatures above 65°C. T. ethanolicus is a promising candidate for thermophilic ethanol fermentations due to the utilization of both pentose and hexose. Although an ethanol balance model in T. ethanolicus has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in ethanol formation have been carried out. To address this issue, we developed a thermostable Cas9-based system for genome editing of T. ethanolicus As a proof of principle, three genes, including the thymidine kinase gene (tdk), acetaldehyde-alcohol dehydrogenase gene (adhE), and redox sensing protein gene (rsp), were chosen as editing targets, and these genes were edited successfully. As a genetic tool, we tested the gene knockout and a small DNA fragment knock-in. After optimization of the transformation strategies, 77% genome-editing efficiency was observed. Furthermore, our in vivo results revealed that redox sensing protein (RSP) plays a more important role in regulation of energy metabolism, including hydrogen production and ethanol formation. The genetic system provides us with an effective strategy to identify genes involved in biosynthesis and energy metabolism.IMPORTANCE Interest in thermophilic microorganisms as emerging metabolic engineering platforms to produce biofuels and chemicals has surged. Thermophilic microbes for biofuels have attracted great attention, due to their tolerance of high temperature and wide range of substrate utilization. On the basis of the biochemical experiments of previous investigation, the formation of ethanol was controlled via transcriptional regulation and influenced by the relevant properties of specific enzymes in T. ethanolicus Thus, there is an urgent need to understand the physiological function of these key enzymes, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Here, we developed a thermostable Cas9-based engineering tool for gene editing in T. ethanolicus The thermostable Cas9-based genome-editing tool may further be applied to metabolically engineer T. ethanolicus to produce biofuels. This genetic system represents an important expansion of the genetic tool box of anaerobic thermophile T. ethanolicus strains.

Harnessing "A Billion Years of Experimentation": The Ongoing Exploration and Exploitation of CRISPR-Cas Immune Systems.

The famed physicist-turned-biologist, Max Delbrück, once remarked that, for physicists, “the field of bacterial viruses is a fine playground for serious children who ask ambitious questions.” Early discoveries in that playground helped establish molecular genetics, and half a century later, biologists delving into the same field have ushered in the era of precision genome engineering. The focus has of course shifted—from bacterial viruses and their mechanisms of infection to the bacterial hosts and their mechanisms of immunity—but it is the very same evolutionary arms race that continues to awe and inspire researchers worldwide. In this review, we explore the remarkable diversity of CRISPR–Cas adaptive immune systems, describe the molecular components that mediate nucleic acid targeting, and outline the use of these RNA-guided machines for biotechnology applications. CRISPR–Cas research has yielded far more than just Cas9-based genome-editing tools, and the wide-reaching, innovative impacts of this fascinating biological playground are sure to be felt for years to come.

Prediction and Validation of Native and Engineered Cas9 Guide Sequences.

Cas9-based technologies rely on native elements of Type II CRISPR-Cas bacterial immune systems, including the trans-activating CRISPR RNA (tracrRNA), CRISPR RNA (crRNA), Cas9 protein, and protospacer-adjacent motif (PAM). The tracrRNA and crRNA form an RNA duplex that guides the Cas9 endonuclease to complementary nucleic acid sequences. Mechanistically, Cas9 initiates interactions by binding to the target PAM sequence and interrogating the target DNA in a 3'-to-5' manner. Complementarity between the guide RNA and the target DNA is key. In natural systems, precise cleavage occurs when the target DNA sequence contains a PAM flanking a sequence homologous to the crRNA spacer sequence. Currently, the majority of commercial Cas9-based genome-editing tools are derived from the Type II CRISPR-Cas system of Streptococcus pyogenes However, a diverse set of Type II CRISPR-Cas systems exist in nature that are potentially valuable for genome engineering applications. Exploitation of these systems requires prediction and validation of both native and engineered dual and single guide RNAs to drive Cas9 functionality. Here, we discuss how to identify the elements of these immune systems to develop next-generation Cas9-based genome-editing tools. We first discuss how to predict tracrRNA sequences and suggest a method for designing single guide RNAs containing only critical structural modules. We then outline how to predict the PAM sequence, which is crucial for determining potential targets for Cas9. Finally, validation of the system elements through transcriptome analysis and interference assays is essential for developing next-generation Cas9-based genome-editing tools.

Guide RNAs: A Glimpse at the Sequences that Drive CRISPR-Cas Systems.

CRISPR-Cas systems provide adaptive immunity in bacteria and archaea. Although there are two main classes of CRISPR-Cas systems defined by gene content, interfering RNA biogenesis, and effector proteins, Type II systems have recently been exploited on a broad scale to develop next-generation genetic engineering and genome-editing tools. Conveniently, Type II systems are streamlined and rely on a single protein, Cas9, and a guide RNA molecule, comprised of a CRISPR RNA (crRNA) and trans-acting CRISPR RNA (tracrRNA), to achieve effective and programmable nucleic acid targeting and cleavage. Currently, most commercially available Cas9-based genome-editing tools use the CRISPR-Cas system from Streptococcus pyogenes (SpyCas9), although many orthogonal Type II systems are available for diverse and multiplexable genome engineering applications. Here, we discuss the biological significance of Type II CRISPR-Cas elements, including the tracrRNA, crRNA, Cas9, and protospacer-adjacent motif (PAM), and look at the native function of these elements to understand how they can be engineered, enhanced, and optimized for genome editing applications. Additionally, we discuss the basis for orthogonal Cas9 and guide RNA systems that would allow researchers to concurrently use multiple Cas9-based systems for different purposes. Understanding the native function of endogenous Type II CRISPR-Cas systems can lead to new Cas9 tool development to expand the genetic manipulation toolbox.