https://backend.orbit.dtu.dk/ws/files/160753786/1_s2.0_S1096717618301502_main.pdf

Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism.

Although polymerase chain reaction (PCR) is the most widely used method for DNA amplification, the requirement of thermocycling limits its non-laboratory applications. Isothermal DNA amplification techniques are hence valuable for on-site diagnostic applications in place of traditional PCR. Here we describe a true isothermal approach for amplifying and detecting double-stranded DNA based on a CRISPR-Cas9-triggered nicking endonuclease-mediated Strand Displacement Amplification method (namely CRISDA). CRISDA takes advantage of the high sensitivity/specificity and unique conformational rearrangements of CRISPR effectors in recognizing the target DNA. In combination with a peptide nucleic acid (PNA) invasion-mediated endpoint measurement, the method exhibits attomolar sensitivity and single-nucleotide specificity in detection of various DNA targets under a complex sample background. Additionally, by integrating the technique with a Cas9-mediated target enrichment approach, CRISDA exhibits sub-attomolar sensitivity. In summary, CRISDA is a powerful isothermal tool for ultrasensitive and specific detection of nucleic acids in point-of-care diagnostics and field analyses.

/pdf/a-crispr-cas9-triggered-strand-displacement-amplification-4e3ifgzumz.pdf

A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection

The ever-expanding set of CRISPR technologies and their programmable RNA-guided nucleases exhibit remarkable flexibility in DNA targeting. However, this flexibility comes with an ever-present constraint: the requirement for a protospacer adjacent motif (PAM) flanking each target. While PAMs play an essential role in self/nonself discrimination by CRISPR-Cas immune systems, this constraint has launched a far-reaching expedition for nucleases with relaxed PAM requirements. Here, we review ongoing efforts toward realizing PAM-free nucleases through natural ortholog mining and protein engineering. We also address potential consequences of fully eliminating PAM recognition and instead propose an alternative nuclease repertoire covering all possible PAM sequences.

CRISPR technologies and the search for the PAM-free nuclease

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) adaptive immune system has been extensively used for gene editing, including gene deletion, insertion, and replacement in bacterial and eukaryotic cells owing to its simple, rapid, and efficient activities in unprecedented resolution. Furthermore, the CRISPR interference (CRISPRi) system including deactivated Cas9 (dCas9) with inactivated endonuclease activity has been further investigated for regulation of the target gene transiently or constitutively, avoiding cell death by disruption of genome. This review discusses the applications of CRISPR/Cas for genome editing in various bacterial systems and their applications. In particular, CRISPR technology has been used for the production of metabolites of high industrial significance, including biochemical, biofuel, and pharmaceutical products/precursors in bacteria. Here, we focus on methods to increase the productivity and yield/titer scan by controlling metabolic flux through individual or combinatorial use of CRISPR/Cas and CRISPRi systems with introduction of synthetic pathway in industrially common bacteria including Escherichia coli. Further, we discuss additional useful applications of the CRISPR/Cas system, including its use in functional genomics.

Applications of CRISPR/Cas System to Bacterial Metabolic Engineering.

According to Darwin’s theory, endless evolution leads to a revolution. One such example is the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)–Cas system, an adaptive immunity system in most archaea and many bacteria. Gene editing technology possesses a crucial potential to dramatically impact miscellaneous areas of life, and CRISPR–Cas represents the most suitable strategy. The system has ignited a revolution in the field of genetic engineering. The ease, precision, affordability of this system is akin to a Midas touch for researchers editing genomes. Undoubtedly, the applications of this system are endless. The CRISPR–Cas system is extensively employed in the treatment of infectious and genetic diseases, in metabolic disorders, in curing cancer, in developing sustainable methods for fuel production and chemicals, in improving the quality and quantity of food crops, and thus in catering to global food demands. Future applications of CRISPR–Cas will provide benefits for everyone and will save countless lives. The technology is evolving rapidly; therefore, an overview of continuous improvement is important. In this review, we aim to elucidate the current state of the CRISPR–Cas revolution in a tailor-made format from its discovery to exciting breakthroughs at the application level and further upcoming trends related to opportunities and challenges including ethical concerns.

Novel CRISPR-Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives.

CRISPR-Cas9 based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here, we identify and characterize ThermoCas9: an RNA-guided DNA-endonuclease from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that ThermoCas9 is active in vitro between 20 C and 70 C, a temperature range much broader than that of the currently used Cas9 orthologues. Additionally, we demonstrate that ThermoCas9 activity at elevated temperatures is strongly associated with the structure of the employed sgRNA. Subsequently, we develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 C in Bacillus smithii and for gene deletion at 37 C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish the first Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

/pdf/characterizing-a-thermostable-cas9-for-bacterial-genome-2a1mdkex1d.pdf

Characterizing a thermostable Cas9 for bacterial genome editing and silencing

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

/pdf/characterizing-a-thermostable-cas9-for-bacterial-genome-4xqnzkvabn.pdf

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Characterizing a thermostable Cas9 for bacterial genome editing and silencing

Characterizing a thermostable Cas9 for bacterial genome editing and silencing