Capillary electrophoresis-based functional genomics screening to discover novel archaeal DNA modifying enzymes

It has been predicted that 30-80% of archaeal genomes remain annotated as hypothetical proteins with no assigned gene function. Further, many archaeal organisms are difficult to grow or are unculturable. To overcome these technical and experimental hurdles, we have developed a high-throughput functional genomics screen that utilizes capillary electrophoresis (CE) to identify nucleic acid modifying enzymes based on activity rather than sequence homology. Here, we describe a functional genomics screening workflow to find DNA modifying enzyme activities encoded by the hyperthermophile Thermococcus kodakarensis ( T. kodakarensis ). Large DNA insert fosmid libraries representing a ∼5-fold average coverage of the T. kodakarensis genome were prepared in E. coli . RNA-seq showed a high fraction (84%) of T. kodakarensis genes were transcribed in E. coli despite differences in promoter structure and translational machinery. Our high-throughput screening workflow used fluorescently labeled DNA substrates directly in heat-treated lysates of fosmid clones with capillary electrophoresis detection of reaction products. Using this method, we identified both a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and a novel AP lyase DNA repair enzyme family (termed 'TK0353') found only in a small subset of Thermococcales. The screening methodology described provides a fast and efficient way to explore the T. kodakarensis genome for a variety of nucleic acid modifying activities and may have implications for similar exploration of enzymes and pathways that underlie core cellular processes in other Archaea. IMPORTANCE This study provides a rapid, simple, high-throughput method to discover novel archaeal nucleic acid modifying enzymes by utilizing a fosmid genomic library, next-generation sequencing and capillary electrophoresis. The method described here provides details necessary to create 384-well fosmid library plates from Thermococcus kodakarensis genomic DNA, sequence 384-well fosmids plates using Illumina next generation sequencing and perform high-throughput functional read-out assays using capillary electrophoresis to identify a variety of nucleic acid modifying activities including DNA cleavage and ligation. We used this approach to identify a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and identify a novel AP lyase enzyme (TK0353) that lacks sequence homology to known nucleic acid modifying enzymes.

Gene Editing in Life Sciences

Gene editing technologies like the CRISPR-Cas9 system have forever changed the way we analyze genetics. As we now have a way to alter genetics, we can now investigate not just how a biological mechanism works, but wonder what we could do to make it better. This paper will discuss how biotechnology and gene editing are integrated into life sciences and biology, where most applications are found. It will be divided into four sections, each addressing how gene editing technology has advanced a field in life science. Firstly, the focus will be on viral detection systems SHERLOCK (Specific High-sensitivity Enzymatic Reporter UnLOCKing) and DETECTR (DNA Endonuclease Targeting CRISPR Trans Reporter), and their importance to the current global pandemic as well as other applications. Then the attention will be diverted into the rapidly popularizing work on gene drives and attempts to drive the evolution of populations of species to benefit mankind. Next, we will discuss the link between biotechnology and species resurrection while discussing ongoing efforts and the ethics of such massive biodiversity shifts. And finally, we discuss possible solutions for the world's hunger crisis using biotechnology with a section on genetically modified crops. I hope to properly explain the growing applications of gene editing by the end of this paper.

A Hyperthermophilic Argonaute From Ferroglobus placidus With Specificity on Guide Binding Pattern

Argonaute proteins (Agos) from thermophilic archaea are involved in several important processes, such as host defense and DNA replication. The catalytic mechanism of Ago from different microbes with great diversity and genome editing potential is attracting increasing attention. Here, we describe an Argonaute from hyperthermophilic Ferroglobus placidus (FpAgo), with a typical DNA-guided DNA endonuclease activity but adopted with only a short guide 15–20 nt length rather than a broad guide selectivity for reported Agos. FpAgo performed the precise cleavage of phosphodiester bonds between 10 and 11 nt on the target strand (counting from the guide strand) guided strictly by 5′-phosphorylated DNA at temperatures ranging from 75 to 99°C. The cleavage activity was regulated by the divalent cations Mn2+, Mg2+, Co2+, and Ni2+. In addition, FpAgo possesses guide/target mismatch tolerance in the seed region but is sensitive to mismatches in the 3′-guide region. Notably, the EMSA assay revealed that the FpAgo-guide-target ternary complex exhibited a stronger binding affinity for short 15 and 16 nt guide DNAs than longer guides. Moreover, we performed structural modeling analyses that implied the unique PAZ domain of FpAgo for 3′-guide recognition and binding to affect guide length specificity. This study broadens our understanding of thermophilic Agos and paves the way for their use in DNA manipulation.

Construction of a G-quadruplex-specific DNA endonuclease

A G-quadruplex-specific DNA endonuclease was constructed by fusing a RHAU G-quadruplex recognition domain with a Fok1 cleavage domain, providing a useful tool for detection of G-quadruplex structures.

Pseudomonas putida MPE, a manganese-dependent endonuclease of the binuclear metallophosphoesterase superfamily, incises single-strand DNA in two orientations to yield a mixture of 3′-PO4 and 3′-OH termini

Pseudomonas putida MPE exemplifies a novel clade of manganese-dependent single-strand DNA endonuclease within the binuclear metallophosphoesterase superfamily. MPE is encoded within a widely conserved DNA repair operon. Via structure-guided mutagenesis, we identify His113 and His81 as essential for DNA nuclease activity, albeit inessential for hydrolysis of bis-p-nitrophenylphosphate. We propose that His113 contacts the scissile phosphodiester and serves as a general acid catalyst to expel the OH leaving group of the product strand. We find that MPE cleaves the 3′ and 5′ single-strands of tailed duplex DNAs and that MPE can sense and incise duplexes at sites of short mismatch bulges and opposite a nick. We show that MPE is an ambidextrous phosphodiesterase capable of hydrolyzing the ssDNA backbone in either orientation to generate a mixture of 3′-OH and 3′-PO4 cleavage products. The directionality of phosphodiester hydrolysis is dictated by the orientation of the water nucleophile vis-à-vis the OH leaving group, which must be near apical for the reaction to proceed. We propose that the MPE active site and metal-bound water nucleophile are invariant and the enzyme can bind the ssDNA productively in opposite orientations.

Rapid and accurate detection of African swine fever virus by DNA endonuclease-targeted CRISPR trans reporter assay

The first case of African swine fever (ASF) outbreak in China was reported in a suburban pig farm in Shenyang in 2018. Since then, the rapid spread and extension of ASF has become the most serious threat for the swine industry. Therefore, rapid and accurate detection of African swine fever virus (ASFV) is essential to provide effective strategies to control the disease. In this study, we developed a rapid and accurate ASFV-detection method based on the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay. By combining recombinase polymerase amplification with CRISPR-Cas12a proteins, the DETECTR assay demonstrated a minimum detection limit of eight copies with no cross reactivity with other swine viruses. Clinical blood samples were detected by DETECTR assay and showed 100% (30/30) agreement with real-time polymerase chain reaction assay. The rapid and accurate detection of ASFV may facilitate timely eradication measures and strict sanitary procedures to control and prevent the spread of ASF.

Prevalence of phase variable epigenetic invertons among host-associated bacteria

Type I restriction-modification (R-M) systems consist of a DNA endonuclease (HsdR, HsdM and HsdS subunits) and methyltransferase (HsdM and HsdS subunits). The hsdS sequences flanked by inverted repeats (referred to as epigenetic invertons) in certain Type I R-M systems undergo invertase-catalyzed inversions. Previous studies in Streptococcus pneumoniae have shown that hsdS inversions within clonal populations produce subpopulations with profound differences in the methylome, cellular physiology and virulence. In this study, we bioinformatically identified six major clades of the tyrosine and serine family invertases homologs from 16 bacterial phyla, which potentially catalyze hsdS inversions in the epigenetic invertons. In particular, the epigenetic invertons are highly enriched in host-associated bacteria. We further verified hsdS inversions in the Type I R-M systems of four representative host-associated bacteria and found that each of the resultant hsdS allelic variants specifies methylation of a unique DNA sequence. In addition, transcriptome analysis revealed that hsdS allelic variations in Enterococcus faecalis exert significant impact on gene expression. These findings indicate that epigenetic switches driven by invertases in the epigenetic invertons broadly operate in the host-associated bacteria, which may broadly contribute to bacterial host adaptation and virulence beyond the role of the Type I R-M systems against phage infection.