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1. Structures, activity and mechanism of the Type IIS restriction endonuclease PaqCI

   https://doi.org/10.1093/nar/gkad228  

   Kennedy, Madison A. ; Hosford, Christopher J. ; Azumaya, Caleigh M. ; Luyten, Yvette A. ; Chen, Minyong ; Morgan, Richard D. ; Stoddard, Barry L. ( March 2023 , Nucleic Acids Research)

   Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in biotechnology for a variety of purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA synthesis applications such as Golden Gate assembly. The most thoroughly studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or similar enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe biochemical analyses of DNA cleavage by the Type IIS PaqCI restriction endonuclease and a series of molecular structures in the presence and absence of multiple bound DNA targets. The enzyme displays a similar tetrameric organization of target recognition domains in the absence or presence of bound substrate, with a significant repositioning of endonuclease domains in a trapped DNA-bound complex that is poised to deliver the first of a series of double-strand breaks. PaqCI and FokI share similar structural mechanisms of DNA cleavage, but considerable differences in their domain organization and quaternary architecture, facilitating comparisons between distinct Type IIS enzymes.

    

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2. High‐efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR /Cas9

   https://doi.org/10.1111/tpj.13446  

   Gil‐Humanes, Javier ; Wang, Yanpeng ; Liang, Zhen ; Shan, Qiwei ; Ozuna, Carmen V. ; Sánchez‐León, Susana ; Baltes, Nicholas J. ; Starker, Colby ; Barro, Francisco ; Gao, Caixia ; et al ( February 2017 , The Plant Journal)

   The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence‐specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved usingAgrobacteriumT‐DNA, biolistics or by stably integrating nuclease‐encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such asNicotinana tabacum(tobacco) andSolanum lycopersicum(tomato), greater than 10‐fold enhancements inGTfrequencies have been achieved usingDNAvirus‐based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundantSSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon‐based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110‐fold increase in expression of a reporter gene relative to non‐replicating controls. Furthermore, replicons carryingCRISPR/Cas9 nucleases and repair templates achievedGTat an endogenousubiquitinlocus at frequencies 12‐fold greater than non‐viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these highGTfrequencies. We also demonstrate gene‐targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with theWDVreplicons, multiplexedGTwithin the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies ofGTusingWDV‐basedDNAreplicons will make it possible to edit complex cereal genomes without the need to integrateGTreagents into the genome.

    

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3. Anacapa Toolkit : An environmental DNA toolkit for processing multilocus metabarcode datasets

   https://doi.org/10.1111/2041-210X.13214  

   Curd, Emily E. ; Gold, Zack ; Kandlikar, Gaurav S. ; Gomer, Jesse ; Ogden, Max ; O'Connell, Taylor ; Pipes, Lenore ; Schweizer, Teia M. ; Rabichow, Laura ; Lin, Meixi ; et al ( June 2019 , Methods in Ecology and Evolution)

   Environmental DNA (eDNA) metabarcoding is a promising method to monitor species and community diversity that is rapid, affordable and non‐invasive. The longstanding needs of the eDNA community are modular informatics tools, comprehensive and customizable reference databases, flexibility across high‐throughput sequencing platforms, fast multilocus metabarcode processing and accurate taxonomic assignment. Improvements in bioinformatics tools make addressing each of these demands within a single toolkit a reality.

   The new modular metabarcode sequence toolkitAnacapa(https://github.com/limey-bean/Anacapa/) addresses the above needs, allowing users to build comprehensive reference databases and assign taxonomy to raw multilocus metabarcode sequence data. A novel aspect ofAnacapais its database building module, “Creating Reference libraries Using eXisting tools” (CRUX), which generates comprehensive reference databases for specific user‐defined metabarcoding loci. TheQuality Control and ASV Parsingmodule sorts and processes multiple metabarcoding loci and processes merged, unmerged and unpaired reads maximizing recovered diversity.DADA2then detects amplicon sequence variants (ASVs) and theAnacapa Classifiermodule aligns these ASVs toCRUX‐generated reference databases usingBowtie2. Lastly, taxonomy is assigned to ASVs with confidence scores using a Bayesian Lowest Common Ancestor (BLCA) method. TheAnacapa Toolkitalso includes anrpackage,ranacapa, for automated results exploration through standard biodiversity statistical analysis.

   Benchmarking tests verify that theAnacapa Toolkiteffectively and efficiently generates comprehensive reference databases that capture taxonomic diversity, and can assign taxonomy to both MiSeq and HiSeq‐length sequence data. We demonstrate the value of theAnacapa Toolkitin assigning taxonomy to seawater eDNA samples collected in southern California.

   TheAnacapa Toolkitimproves the functionality of eDNA and streamlines biodiversity assessment and management by generating metabarcode specific databases, processing multilocus data, retaining a larger proportion of sequencing reads and expanding non‐traditional eDNA targets. All the components of theAnacapa Toolkitare open and available in a virtual container to ease installation.

    

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4. GoldBricks: an improved cloning strategy that combines features of Golden Gate and BioBricks for better efficiency and usability

   https://doi.org/10.1093/synbio/ysab032  

   Chaudhari, Vishalsingh R. ; Hanson, Maureen R. ( November 2021 , Synthetic Biology)

   With increasing complexity of expression studies and the repertoire of characterized sequences, combinatorial cloning has become a common necessity. Techniques like BioBricks and Golden Gate aim to standardize and speed up the process of cloning large constructs while enabling sharing of resources. The BioBricks format provides a simplified and flexible approach to endless assembly with a compact library and useful intermediates but is a slow process, joining only two parts in a cycle. Golden Gate improves upon the speed with use of Type IIS enzymes and joins several parts in a cycle but requires a larger library of parts and logistical inefficiencies scale up significantly in the multigene format. We present here a method that provides improvement over these techniques by combining their features. By using Type IIS enzymes in a format like BioBricks, we have enabled a faster and efficient assembly with reduced scarring, which performs at a similarly fast pace as Golden Gate, but significantly reduces library size and user input. Additionally, this method enables faster assembly of operon-style constructs, a feature requiring extensive workaround in Golden Gate. Our format allows such inclusions resulting in faster and more efficient assembly.

    

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5. Effective design principles for leakless strand displacement systems

   https://doi.org/10.1073/pnas.1806859115  

   Wang, Boya ; Thachuk, Chris ; Ellington, Andrew D. ; Winfree, Erik ; Soloveichik, David ( December 2018 , Proceedings of the National Academy of Sciences)

   Artificially designed molecular systems with programmable behaviors have become a valuable tool in chemistry, biology, material science, and medicine. Although information processing in biological regulatory pathways is remarkably robust to error, it remains a challenge to design molecular systems that are similarly robust. With functionality determined entirely by secondary structure of DNA, strand displacement has emerged as a uniquely versatile building block for cell-free biochemical networks. Here, we experimentally investigate a design principle to reduce undesired triggering in the absence of input (leak), a side reaction that critically reduces sensitivity and disrupts the behavior of strand displacement cascades. Inspired by error correction methods exploiting redundancy in electrical engineering, we ensure a higher-energy penalty to leak via logical redundancy. Our design strategy is, in principle, capable of reducing leak to arbitrarily low levels, and we experimentally test two levels of leak reduction for a core “translator” component that converts a signal of one sequence into that of another. We show that the leak was not measurable in the high-redundancy scheme, even for concentrations that are up to 100 times larger than typical. Beyond a single translator, we constructed a fast and low-leak translator cascade of nine strand displacement steps and a logic OR gate circuit consisting of 10 translators, showing that our design principle can be used to effectively reduce leak in more complex chemical systems. 

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