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Gene Disruption of Honey Bee Trypanosomatid Parasite, Lotmaria passim, by CRISPR/Cas9 System.
Two trypanosomatid species, Lotmaria passim and Crithidia mellificae, have been shown to parasitize honey bees to date. L. passim appears to be more prevalent than C. mellificae and specifically infects the honey bee hindgut. Although the genomic DNA has been sequenced, the effects of infection on honey bee health and colony are poorly understood. To identify the genes that are important for infecting honey bees and to understand their functions, we applied the CRISPR/Cas9 system to establish a method to manipulate L. passim genes. By electroporation of plasmid DNA and subsequent selection by drug, we first established an L. passim clone expressing tdTomato or Cas9. We also successfully disrupted the endogenous miltefosine transporter and tyrosine aminotransferase genes by replacement with drug (hygromycin) resistant gene using the CRISPR/Cas9-induced homology-directed repair pathway. The L. passim clone expressing fluorescent marker, as well as the simple method for editing specific genes, could become useful approaches to understand the underlying mechanisms of honey bee-trypanosomatid parasite interactions.
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An Introduction to Methods for Discovery and Functional Analysis of MicroRNAs in Plants.
MicroRNAs play important roles in posttranscriptional regulation of plant development, metabolism, and abiotic stress responses. The recent generation of massive amounts of small RNA sequence data, along with development of bioinformatic tools to identify miRNAs and their mRNA targets, has led to an explosion of newly identified putative miRNAs in plants. Genome editing techniques like CRISPR-Cas9 will allow us to study the biological role of these potential novel miRNAs by efficiently targeting both the miRNA and its mRNA target. In this chapter, we review bioinformatic tools and experimental methods for the identification and functional characterization of miRNAs and their target mRNAs in plants.
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Plasmid-Free CRISPR/Cpf1 Genome Editing With In Vivo T7 RNA Polymerase-Transcribed CRISPR RNA From Short Double-Stranded DNA.
Plasmids are commonly employed in the delivery of clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) components for genome editing. However, the absence of heritable plasmids in numerous organisms limits the development of CRISPR/Cas genome editing tools. Moreover, cumbersome procedures for plasmid construction and curing render genome editing time-consuming. In this study, we developed a plasmid-free CRISPR/Cpf1 genome editing system for Saccharomyces cerevisiae and Starmerella bombicola. This system leveraged integrative expression of the Cpf1 nuclease and T7 RNA polymerase (T7RNAP), as well as the delivery of linear fragments including (i) a marker cassette for integration and selection, (ii) short double-stranded DNA (crDNA) for in vivo transcription of crRNA by T7RNAP, and (iii) donor DNA for homology-directed repair. We demonstrated that this editing system enabled efficient multiplexed and iterative genome editing without the need for marker recycling and plasmid curing. The use of short crDNA (87 bp) and donor DNA (≤ 120 bp), both readily prepared from ordered oligonucleotides via annealing or overlap extension, dramatically simplified the editing process. Successful implementation in S. bombicola, which lacks heritable plasmids for genetic engineering, highlighted the potential of this approach especially for genome engineering of genetically intractable organisms in a plasmid-free way.
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Diverse viral cas genes antagonize CRISPR immunity.
Prokaryotic CRISPR-Cas immunity is subverted by anti-CRISPRs (Acrs), which inhibit Cas protein activities when expressed during the phage lytic cycle or from resident prophages or plasmids1. Acrs often bind to specific cognate Cas proteins, and hence inhibition is typically limited to a single CRISPR-Cas subtype2. Furthermore, although acr genes are frequently organized together in phage-associated gene clusters3, how such inhibitors initially evolve has remained unclear. Here we investigated the Acr content and inhibition specificity of diverse Listeria isolates, which naturally harbour four CRISPR-Cas systems (types I-B, II-A, II-C and VI-A). We observed widespread antagonism of CRISPR, which we traced to 11 previously unknown and 4 known acr gene families encoded by endogenous mobile elements. Among these were two Acrs that possess sequence homology to type I-B Cas proteins, one of which assembles into a defective interference complex. Surprisingly, an additional type I-B Cas homologue did not affect type I immunity, but instead inhibited the RNA-targeting type VI CRISPR system by means of CRISPR RNA (crRNA) degradation. By probing viral sequence databases, we detected abundant orphan cas genes located within putative anti-defence gene clusters. Among them, we verified the activity of a particularly broad-spectrum cas3 homologue that inhibits type I-B, II-A and VI-A CRISPR immunity. Our observations provide direct evidence of Acr evolution by cas gene co-option, and new genes with potential for broad-spectrum control of genome editing technologies.
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Governing Human Germline Editing Through Patent Law.
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Tracking and Mapping the Use of CRISPR-Cas Systems for Research in China.
Scientists have floated the idea of a "Sputnik 2.0" technological race between the People's Republic of China (PRC) and the United States of America in CRISPR-based genome editing research. This quantitative analysis of articles published between 2010 and 2020 shows that research centers based in the PRC have succeeded in making CRISPR-based genome editing a standard tool. A corpus of 18,863 academic documents containing the acronym CRISPR in their abstract shows that although PRC-based research institutions were slower to start publishing on CRISPR, they have now outpaced the publication rate of institutions located in the European Union (EU). While U.S.-based institutions have kept their leading position in basic research, PRC-based research has become momentous in agriculture-related fields. This corpus hence illustrates how deeply the international landscape of life sciences research has shifted since the Human Genome Project, mostly to the PRC's advantage.
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CRISPR-based functional genomics for schistosomes and related flatworms.
CRISPR genome editing is actively used for schistosomes and other flukes. The ability to genetically manipulate these flatworms enables deeper investigation of their (patho)biological nature. CRISPR gene knockout (KO) demonstrated that a liver fluke growth mediator contributes to disease progression. Genome safe harbor sites have been predicted in Schistosoma mansoni and targeted for transgene insertion. CRISPR-based diagnosis has been demonstrated for infection with schistosomes and Opisthorchis viverrini. This review charts the progress, and the state of play, and posits salient questions for the field to address. Derivation of heritably transgenic loss-of-function or gain-of-function lines is the next milestone.
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Predicting and visualizing features of CRISPR-Cas systems.
Pervasive application of CRISPR-Cas systems in genome editing has prompted an increase in both interest and necessity to further elucidate existing systems as well as discover putative novel systems. The ubiquity and power of current computational platforms have made in silico approaches to CRISPR-Cas identification and characterization accessible to a wider audience and increasingly amenable for processing extensive data sets. Here, we describe in silico methods for predicting and visualizing notable features of CRISPR-Cas systems, including Cas domain determination, CRISPR array visualization, and inference of the protospacer-adjacent motif. The efficiency of these tools enables rapid exploration of CRISPR-Cas diversity across prokaryotic genomes and supports scalable analysis of large genomic data sets.
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High-Throughput Functional Genomics for Energy Production.
Functional genomics remains a foundational field for establishing genotype-phenotype relationships that enable strain engineering. High-throughput (HTP) methods accelerate the Design-Build-Test-Learn cycle that currently drives synthetic biology towards a forward engineering future. Trackable mutagenesis techniques including transposon insertion sequencing and CRISPR-Cas-mediated genome editing allow for rapid fitness profiling of a collection, or library, of mutants to discover beneficial mutations. Due to the relative speed of these experiments compared to adaptive evolution experiments, iterative rounds of mutagenesis can be implemented for next-generation metabolic engineering efforts to design complex production and tolerance phenotypes. Additionally, the expansion of these mutagenesis techniques to novel bacteria are opening up industrial microbes that show promise for establishing a bio-based economy.
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Metabolic engineering of Bacillus subtilis for l-valine overproduction.
Bacillus subtilis has been commonly applied to industrial enzyme production due to its genetic tractability, "generally recognized as safe (GRAS)" status, and robust growth characteristics. In spite of its ideal attributes as a biomanufacturing platform, B. subtilis has seen limited use in the production of other value-added biochemicals. Here, we report the derivation of engineered strains of B. subtilis for l-valine overproduction using our recently developed CRISPR (clustered regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated [protein] 9) toolkit. We first manipulate the native l-valine biosynthetic pathway by relieving transcriptional and allosteric regulation, resulting in a >14-fold increase in the l-valine titer, compared to the wild-type strain. We subsequently identify and eliminate factors limiting l-valine overproduction, specifically increasing pyruvate availability and blocking the competing l-leucine and l-isoleucine biosynthetic pathways. By inactivating (a) pdhA, encoding the E1α subunit of the pyruvate dehydrogenase complex, to increase the intracellular pyruvate pool, and (b) leuA and ilvA, respectively encoding 2-isopropylmalate synthase and l-threonine dehydratase, to abolish the competing pathways, the l-valine titer reached 4.61 g/L in shake flask cultures. Our engineered l-valine-overproducing strains of B. subtilis are devoid of plasmids and do not sporulate due to the inactivation of sigF, encoding the sporulation-specific transcription factor σ F , making them attractive for large-scale l-valine production. However, acetate dissimilation was identified as limiting l-valine overproduction in ΔpdhA B. subtilis strains, and improving acetate dissimilation or identifying alternate modes of increasing pyruvate pools to enhance l-valine-overproduction should be explored.
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