Modulating CRISPR-Cas genome editing using guide-complementary DNA oligonucleotides

CRISPR-Cas has revolutionized genome editing and has a great potential for applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. Here we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides down-regulate Cas9 activity in human cells, reducing both on and off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: the length of the complementary region, and the presence of a PAM-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.

Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus

Gene therapy would benefit from a miniature CRISPR system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in eukaryotic cells. One of the most compact CRISPR-associated nucleases yet discovered is the archaeal Un1Cas12f1. However, Un1Cas12f1 and its variants have very low activity in eukaryotic cells. In the present study, we redesigned the natural guide RNA of Un1Cas12f1 at five sites: the 5′ terminus of the trans-activating CRISPR RNA (tracrRNA), the tracrRNA–crRNA complementary region, a penta(uridinylate) sequence, the 3′ terminus of the crRNA and a disordered stem 2 region in the tracrRNA. These optimizations synergistically increased the average indel frequency by 867-fold. The optimized Un1Cas12f1 system enabled efficient, specific genome editing in human cells when delivered by plasmid vectors, PCR amplicons and AAV. As Un1Cas12f1 cleaves outside the protospacer, it can be used to create large deletions efficiently. The engineered Un1Cas12f1 system showed efficiency comparable to that of SpCas9 and specificity similar to that of AsCas12a.

How Much Is Enough? First Steps to a Social Ecology of the Pergamon Microregion

In this study, we present a transparent and reproducible approach to model agricultural production with respect to environmental characteristics and available labour. Our research focuses on the city of Pergamon and its surroundings, with an emphasis on the transition between the Hellenistic and Roman Imperial Period, where widespread demographic changes took place. We investigated the degree of local self-sufficiency using different concepts of a city’s complementary region. Using simple topographic derivatives, we derive a measure of environmental suitability that we translate into a carrying capacity index. Our results show that workforce was not a limiting factor for local self-sufficiency. However, environmental carrying capacity may have been limiting in a scenario with a large population. An active investment into the environment, e.g., by the construction of terraces, could have helped to increase the degree of self-sufficiency. Future research should investigate the level of resilience of such a coupled socio-ecological system in relation to environmental and socio-cultural dynamics.

1654. Evaluation of a rapid detection method of clarithromycin resistance genes in Mycobacterium avium using the Amplification Refractory Mutation System-Loop-Mediated Isothermal Amplification method

Background Clarithromycin (CLR) is the key drug in multidrug therapy for Mycobacterium avium complex (MAC) diseases and the only drug for which drug susceptibility is correlated with a clinical response in these diseases. In the case of CLR-resistant MAC, a point mutation is present at either position 2058 or 2059 of the peptidyl transferase active center in the domain V region of 23S rRNA at the macrolide binding site. Using conventional investigation, we clarified the correlation between drug susceptibility testing and mutation of drug resistance genes. In this study, we adapted a rapid detection method using the amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) to identify a mutation in the 23S rRNA gene in M. avium isolates (Figure 1). Furthermore, we evaluated the usefulness as point-of-care testing (POCT) technology using clinical isolates. Figure 1. The designs of CLR resistance A2058G mutant-type mismatch primers used for the ARMS-LAMP assay. a) A strand-displacing DNA polymerase extends the DNA from FIP while separating from the DNA chain. The primer F3 binds to its complementary region on the DNA to displace the newly synthesized DNA. An analogous reaction is performed by BIP and B3. α (α = A, wild type; G, A2058G) and β (β = A, wild type; C, A2058G) are indicated by the point mutation at position 2058 of the 23S rRNA gene. The bold area indicates the mismatched base C (cytosine). b) The synthesized DNA self-anneals because of the complementary region at both ends and forms ‘dumbbell’ structures. c) After repeated rounds, a complementary region on the same chain is amplified. Methods Primers for ARMS-LAMP were designed using PrimerExplorerV5 software based on the nucleotide sequence data for 23S rRNA in M. avium strain 104 (Figure 2). Using the minimum inhibitory concentration of CLR, drug susceptibility was determined for 18 clinical M. avium isolates. Of these, eight CLR-susceptible and 10 CLR-resistant strains were analyzed by sequencing the 23S rRNA gene and ARMS-LAMP. Figure 2. Alignment of the nucleotide sequences including the domain V region of 23S rRNA at the macrolide binding site. The constructed LAMP primer sets are shown in solid boxes (forward primers, F1-3) and dashed boxes (backward primers, B1-3). The bold area indicates the point mutation at position 2058 or 2059 of the 23S rRNA gene. Results Sequence analysis revealed that all eight CLR-sensitive strains tested were wild type, whereas all 10 CLR-resistant strains were mutants. Using ARMS-LAMP, no amplification with the mutant-type mismatch primer sets (MTPS) was observed in the eight wild-type strains, but amplification was observed with MTPS in the 10 mutant strains (Table 1). Table 1. MICs of CLR and results of ARMS–LAMP using Mycobacterium avium isolates. Conclusion The developed rapid detection method for the CLR resistance gene using ARMS-LAMP can determine drug resistance in a few hours without the need for special equipment. ARMS-LAMP may be a new clinically beneficial POCT technology for examination that is novel and extremely practical. Disclosures All Authors: No reported disclosures

Structural Biology of the Enterovirus Replication-Linked 5′-Cloverleaf RNA and Associated Virus Proteins

SUMMARY Although enteroviruses are associated with a wide variety of diseases and conditions, their mode of replication is well conserved. Their genome is carried as a single, positive-sense RNA strand. At the 5′ end of the strand is an approximately 90-nucleotide self-complementary region called the 5′ cloverleaf, or the oriL. This noncoding region serves as a platform upon which host and virus proteins, including the 3B, 3C, and 3D virus proteins, assemble in order to initiate replication of a negative-sense RNA strand. The negative strand in turn serves as a template for synthesis of multiple positive-sense RNA strands. Building on structural studies of individual RNA stem-loops, the structure of the intact 5′ cloverleaf from rhinovirus has recently been determined via nuclear magnetic resonance/small-angle X-ray scattering (NMR/SAXS)-based methods, while structures have also been determined for enterovirus 3A, 3B, 3C, and 3D proteins. Analysis of these structures, together with structural and modeling studies of interactions between host and virus proteins and RNA, has begun to provide insight into the enterovirus replication mechanism and the potential to inhibit replication by blocking these interactions.

DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates

The Dictyostelium Intermediate Repeat Sequence 1 (DIRS-1) is the name-giving member of the DIRS order of tyrosine recombinase retrotransposons. In Dictyostelium discoideum, DIRS-1 is highly amplified and enriched in heterochromatic centromers of the D. discoideum genome. We show here that DIRS-1 it tightly controlled by the D. discoideum RNA interference machinery and is only mobilized in mutants lacking either the RNA dependent RNA polymerase RrpC or the Argonaute protein AgnA. DIRS retrotransposons contain an internal complementary region (ICR) that is thought to be required to reconstitute a full-length element from incomplete RNA transcripts. Using different versions of D. discoideum DIRS-1 equipped with retrotransposition marker genes, we show experimentally that the ICR is in fact essential to complete retrotransposition. We further show that DIRS-1 produces a mixture of single-stranded, mostly linear extrachromosomal cDNA intermediates. If this cDNA is isolated and transformed into D. discoideum cells, it can be used by DIRS-1 proteins to complete productive retrotransposition. This work provides the first experimental evidence to propose a general retrotransposition mechanism of the class of DIRS like tyrosine recombinase retrotransposons.

EMBEDDING OF METRIC GRAPHS ON HYPERBOLIC SURFACES

An embedding of a metric graph $(G,d)$ on a closed hyperbolic surface is essential if each complementary region has a negative Euler characteristic. We show, by construction, that given any metric graph, its metric can be rescaled so that it admits an essential and isometric embedding on a closed hyperbolic surface. The essential genus $g_{e}(G)$ of $(G,d)$ is the lowest genus of a surface on which such an embedding is possible. We establish a formula to compute $g_{e}(G)$ and show that, for every integer $g\geq g_{e}(G)$ , there is an embedding of $(G,d)$ (possibly after a rescaling of $d$ ) on a surface of genus $g$ . Next, we study minimal embeddings where each complementary region has Euler characteristic $-1$ . The maximum essential genus $g_{e}^{\max }(G)$ of $(G,d)$ is the largest genus of a surface on which the graph is minimally embedded. We describe a method for an essential embedding of $(G,d)$ , where $g_{e}(G)$ and $g_{e}^{\max }(G)$ are realised.

A Minigenome Study of Hazara Nairovirus Genomic Promoters

ABSTRACTHazara nairovirus (HAZV) is a trisegmented RNA virus most closely related to Crimean-Congo hemorrhagic fever virus (CCHFV) in the orderBunyavirales. The terminal roughly 20 nucleotides (nt) of its genome ends are highly complementary, similar to those of other segmented negative-strand RNA viruses (sNSV), and act as promoters for RNA synthesis. These promoters contain two elements: the extreme termini of both strands (promoter element 1 [PE1]) are conserved and virus specific and are found bound to separate sites on the polymerase surface in crystal structures of promoter-polymerase complexes. The following sequences (PE2) are segment specific, with the potential to form double-stranded RNA (dsRNA), and the latter aspect is also important for promoter activity. Nairovirus genome promoters differ from those of peribunyaviruses and arenaviruses in that they contain a short single-stranded region between the two regions of complementarity. Using a HAZV minigenome system, we found the single-stranded nature of this region, as well as the potential of the following sequence to form dsRNA, is essential for reporter gene expression. Most unexpectedly, the sequence of the PE2 dsRNA appears to be equally important for promoter activity. These differences in sNSV PE2 promoter elements are discussed in light of our current understanding of the initiation of RNA synthesis.IMPORTANCEA minigenome system for HAZV, closely related to CCHFV, was used to study its genome replication. HAZV genome ends, like those of other sNSV, such as peribunyaviruses and arenaviruses, are highly complementary and serve as promoters for genome synthesis. These promoters are composed of two elements: the extreme termini of both 3′ and 5′ strands that are initially bound to separate sites on the polymerase surface in a sequence-specific fashion and the following sequences with the potential to anneal but whose sequence is not important. Nairovirus promoters differ from the other sNSV cited in that they contain a short single-stranded RNA (ssRNA) region between the two elements. The single-stranded nature of this region is an essential element of the promoter, whereas its sequence is unimportant. The sequence of the following complementary region is unexpectedly also important, a possible rare example of sequence-specific dsRNA recognition.

On the Watercolor Illusion

The watercolor illusion is a long-range assimilative spread of color emanating from a thin colored line running contiguous to a darker chromatic contour and imparting a figure-ground effect across a large area. The watercolored figure appears evenly colored by an opaque light veil of chromatic tint (coloration effect), with a clear surface color property spreading from the lighter edges. At the same time, the watercolored figure manifests a strong figure-ground organization and a solid figural appearance comparable to a rounded surface segregated in depth which extends out from the flat surface. The complementary region appears as a hole or empty space. The phenomenal properties of coloration and figure-ground effects and their relationship are described and demonstrated. The watercolor illusion and its main effects are discussed in the light of parallel mechanisms. Boundary and surface dynamics are processed by the boundary contour system and by the feature contour system.

A porphycene-DNA hybrid and its DNA-templated interactions with a porphyrin

A porphycene with a hydroxyethyl side chain was coupled to the 3′-terminus of an oligodeoxynucleotide via solid-phase synthesis. The resulting porphycene-DNA hybrid binds to a complementary region of a DNA target strand with greater affinity than the unmodified control oligonucleotide, resulting in an increase of the UV-melting point of 12.7 °C. Duplex formation is accompanied by an increase in porphycene fluorescence at 640 nm by 18%. When a tetrakis(p-hydroxyphenyl)porphyrin appended to the 5′-terminus of another DNA-strand is brought into close proximity of the porphycene by hybridizing it to the downstream-region of the template strand, 94% of the porphycene fluorescence is quenched. By quenching each others fluorescence to different degrees, porphycene and porphyrin, together, report on local DNA structure in a fashion reminiscent of that of molecular beacons. The introduction of porphycenes and the porphycene-porphyrin "two hybrid system'' to DNA-based structuring may open up new avenues to designed functional materials.