Coping with ageing in rural Australia.

OBJECTIVE: Ageing is a time of change that might involve financial, health and social losses. To maintain well-being, older people need to engage a range of resources to cope with these losses. However, national policies mainly focus on financial resources. This study used Folkman and Lazarus's transactional theory to identify coping methods engaged by older adults living in three rural communities. DESIGN: A qualitative research design was undertaken using an ethnographic case study approach. SETTING: Three rural communities within northern Australia. PARTICIPANTS: Older Australians aged 65 or over, living in their own homes. MAIN OUTCOME MEASURES: Exploration of techniques that older adults use to cope with ageing (including both problem-focused and emotion-focused strategies). RESULTS: People in rural areas planned their ageing journey using both problem-focused and emotion-focused coping strategies, to deal with the uncertainties of ageing. When participants could control the event, problem-focused coping strategies were mainly such as used seeking social support and planful problem-solving. Conversely, emotion-focused strategies were used to deal with uncertainty and emotive issues such as health decline, and the possibility of needing future care. CONCLUSION: There is a need for health community workers to encourage older people to consider initiating a discussion of future care needs with their social network. This is of particular importance in rural areas, which have larger numbers of older residents and limited resources to support ageing in place.

Conjugation and Evaluation of Triazole-Linked Single Guide RNA for CRISPR-Cas9 Gene Editing.

The CRISPR-Cas9 gene editing system requires Cas9 endonuclease and guide RNAs (either the natural dual RNA consisting of crRNA and tracrRNA or a chimeric single guide RNA) that direct site-specific double-stranded DNA cleavage. This communication describes a click ligation approach that uses alkyne-azide cycloaddition to generate a triazole-linked single guide RNA (sgRNA). The conjugated sgRNA shows efficient and comparable genome editing activity to natural dual RNA and unmodified sgRNA constructs.

A Facile, Transfection-Free Approach to siRNA Delivery in In Vitro 3D Spheroid Models.

Cell culture has long been essential for preclinical modeling of human development and disease. However, conventional two-dimensional (2D) cell culture fails to faithfully model the complexity found in vivo, and novel drug candidates that show promising results in 2D models often do not translate to the clinic. More recently, three-dimensional (3D) cell culture models have gained popularity owing to their greater physiological relevance to in vivo biology. In particular, 3D spheroid models are becoming widely used due to their ability to mimic solid tumors, both in architecture and gradation of nutrients distributed from the outer, proliferative layers into the inner, quiescent layers of cells. Similar to in vivo tumors, cell lines grown in 3D spheroid models tend to be more resistant to antitumor drug treatments than their 2D cultured counterparts, though distinct signaling pathways and gene targets conferring this resistance have yet to be fully explored. RNA interference (RNAi) is an effective tool to elucidate gene function and discover novel druggable targets in 2D models; however, only a few studies have successfully performed RNAi in complex 3D models to date. Here, we demonstrate efficient RNAi-mediated knockdown using "transfection-free" Dharmacon Accell siRNAs in three spheroid culture models, in the presence or absence of the extracellular matrix. This methodology has the potential to be scaled up for complex arrayed screening experiments, which may aid in the identification of novel druggable targets with greater clinical relevance than those identified in 2D experiments. © 2024 Dharmacon, Inc. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 3D spheroids in matrix-free ULA plates Alternate Protocol 1: Generation of Matrigel matrix-embedded 3D spheroids Alternate Protocol 2: Generation of GrowDex hydrogel-embedded 3D spheroids Basic Protocol 2: Delivery of siRNA and collection of matrix-free 3D spheroids Alternate Protocol 3: Delivery of siRNA and collection of matrix-embedded spheroids Basic Protocol 3: RNA and protein extraction from spheroids for characterization of gene knockdown.

Experimental validation of the importance of seed complement frequency to siRNA specificity.

Pairing between the hexamer seed region of a small interfering RNA (siRNA) guide strand (nucleotides 2-7) and complementary sequences in the 3' UTR of mature transcripts has been implicated as an important element in off-target gene regulation and false positive phenotypes. To better understand the association between seed sequences and off-target profiles we performed an analysis of all possible (4096) hexamers and identified a nonuniform distribution of hexamer frequencies across the 3' UTR transcriptome. Subsequent microarray analysis of cells transfected with siRNAs having seeds with low, medium, or high seed complement frequencies (SCFs) revealed that duplexes with low SCFs generally induced fewer off-targets and off-target phenotypes than molecules with more abundant 3' UTR complements. These findings provide the first experimentally validated strategy for designing siRNAs with enhanced specificity and allow for more accurate interpretation of high throughput screening data generated with existing siRNA/shRNA collections.

CRISPR-mediated transcriptional activation with synthetic guide RNA.

The CRISPR-Cas9 system has been adapted for transcriptional activation (CRISPRa) and several second-generation CRISPRa systems (including VPR, SunTag, and SAM) have been developed to recruit different transcriptional activators to a deactivated Cas9, which is guided to a transcriptional start site via base complementarity with a target guide RNA. Multiple studies have shown the benefit of CRISPRa using plasmid or lentiviral expressed guide RNA, but the use of synthetic guide RNA has not been reported. Here we demonstrate the effective use of synthetic guide RNA for gene activation via CRISPRa. CRISPRa crRNA may be used with a canonical tracrRNA using the VPR or SunTag activation systems or with an extended tracrRNA containing an aptamer sequence for the SAM system. Transcriptional activation with synthetic crRNA:tracrRNA is comparable to activation achieved with expression vectors and combining several crRNA sequences targeting the same gene can enhance transcriptional activation. The use of synthetic crRNA is also ideal for simultaneous activation of multiple genes or use with dCas9-VPR mRNA when viral transduction is not feasible. Here, we perform a proof-of-principle arrayed screen using a CRISPRa crRNA library consisting of 153 cytokine receptor targets to identify regulators of IL-6 cytokine secretion. Together, these results demonstrate the suitability of synthetic CRISPRa guide RNA for high throughput, arrayed screening applications which allow for more complex phenotypic readouts to complement viability and drug resistance assays typically used in a pooled screening format.

ErCas12a CRISPR-MAD7 for Model Generation in Human Cells, Mice, and Rats.

MAD7 is an engineered class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) system isolated from Eubacterium rectale. Analogous to Cas9, it is an RNA-guided nuclease with demonstrated gene editing activity in Escherichia coli and yeast cells. Here, we report that MAD7 is capable of generating indels and fluorescent gene tagging of endogenous genes in human HCT116 and U2OS cancer cell lines, respectively. In addition, MAD7 is highly proficient in generating indels, small DNA insertions (23 bases), and larger integrations ranging from 1 to 14 kb in size in mouse and rat embryos, resulting in live-born transgenic animals. Due to the different protospacer adjacent motif requirement, small-guide RNA, and highly efficient targeted gene disruption and insertions, MAD7 can expand the CRISPR toolbox for genome enginnering across different systems and model organisms.

Should I Stay or Go: Rural Ageing, a Time for Reflection.

(1) Background: Studies have shown that older people prefer to continue living in their own home and community as they age; however this is dependent upon available services and social support. In Australia about two thirds of people will age at home. The Australian Government provides home care packages to support ageing in place yet in rural areas not all services are available. The lack of employment opportunities in rural areas often results in family residing at a distance reducing available social support. This study aims to evaluate informal social support and its influence on ageing in place amongst older people in three Australian rural communities in Australia. (2) Methods: A multiple embedded case study was undertaken in three diverse rural communities. Eleven older rural residents ageing in place aged 65+ were interviewed about their ageing experience and plans for their future in the light of available social support along with 15 members of their social networks. Social networks were then visually depicted with the use of ecomaps and network members were interviewed. (3) Results show that kin and non-kin social networks support ageing in place however ageing is a time of change and reflection. (4) Conclusions: There is a need for more discussion within these networks when it comes to future planning.

Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif recognition.

While the CRISPR-Cas9 system from S. pyogenes is a powerful genome engineering tool, additional programmed nucleases would enable added flexibility in targeting space and multiplexing. Here, we characterized a CRISPR-Cas9 system from L. gasseri and found that it has modest activity in a cell-free lysate assay but no activity in mammalian cells even when altering promoter, position of tag sequences and NLS, and length of crRNA:tracrRNA. In the lysate assay we tested over 400 sequential crRNA target sequences and found that the Lga Cas9 PAM is NNGA/NDRA, different than NTAA predicted from the native bacterial host. In addition, we found multiple instances of consecutive crRNA target sites, indicating flexibility in either PAM sequence or distance from the crRNA target site. This work highlights the need for characterization of new CRISPR systems and highlights the non-triviality of porting them into eukaryotes as gene editing tools.

High-content analysis screening for cell cycle regulators using arrayed synthetic crRNA libraries.

The CRISPR-Cas9 system has been utilized for large-scale, loss-of-function screens mainly using lentiviral pooled formats and cell-survival phenotypic assays. Screening in an arrayed format expands the types of phenotypic readouts that can be used to now include high-content, morphology-based assays, and with the recent availability of synthetic crRNA libraries, new studies are emerging. Here, we use a cell cycle reporter cell line to perform an arrayed, synthetic crRNA:tracrRNA screen targeting 169 genes (>600 crRNAs) and used high content analysis (HCA) to identify genes that regulate the cell cycle. Seven parameters were used to classify cells into cell cycle categories and multiple parameters were combined using a new analysis technique to identify hits. Comprehensive hit follow-up experiments included target gene expression analysis, confirmation of DNA insertions/deletions, and validation with orthogonal reagents. Our results show that most hits had three or more independent crRNAs per gene that demonstrated a phenotype with consistent individual parameters, indicating that our screen produced high-confidence hits with low off-target effects and allowed us to identify hits with more subtle phenotypes. The results of our screen demonstrate the power of using arrayed, synthetic crRNAs for functional phenotypic screening using multiparameter HCA assays.

Delineation of the high-affinity single-stranded telomeric DNA-binding domain of Saccharomyces cerevisiae Cdc13.

Cdc13 is an essential protein from Saccharomyces cerevisiae that caps telomeres by protecting the C-rich telomeric DNA strand from degradation and facilitates telomeric DNA replication by telomerase. In vitro, Cdc13 binds TG-rich single-stranded telomeric DNA with high affinity and specificity. A previously identified domain of Cdc13 encompassing amino acids 451-694 (the 451-694 DBD) retains the single-stranded DNA-binding properties of the full-length protein; however, this domain contains a large unfolded region identified in heteronuclear NMR experiments. Trypsin digestion and MALDI mass spectrometry were used to identify the minimal DNA-binding domain (the 497-694 DBD) necessary and sufficient for full DNA-binding activity. This domain was completely folded, and the N-terminal unfolded region removed was shown to be dispensable for function. Using affinity photocrosslinking to site-specifically modified telomeric single-stranded DNA, the 497-694 DBD was shown to contact the entire 11mer required for high-affinity binding. Intriguingly, both domains bound single-stranded telomeric DNA with much greater affinity than the full-length protein. The full-length protein exhibited the same rate of dissociation as both domains, however, indicating that the full-length protein contains a region that inhibits association with single-stranded telomeric DNA.

Structural basis for telomeric single-stranded DNA recognition by yeast Cdc13.

The essential budding yeast telomere-binding protein Cdc13 is required for telomere replication and end protection. Cdc13 specifically binds telomeric, single-stranded DNA (ssDNA) 3' overhangs with high affinity using an OB-fold domain. We have determined the high-resolution solution structure of the Cdc13 DNA-binding domain (DBD) complexed with a cognate telomeric ssDNA. The ssDNA wraps around one entire face of the Cdc13-DBD OB-fold in an extended, irregular conformation. Recognition of the ssDNA bases occurs primarily through aromatic, basic, and hydrophobic amino acid residues, the majority of which are evolutionarily conserved among budding yeast species and contribute significantly to the energetics of binding. Contacting five of 11 ssDNA nucleotides, the large, ordered beta2-beta3 loop is crucial for complex formation and is a unique elaboration on the binding mode commonly observed in OB-fold proteins. The sequence-specific Cdc13-DBD/ssDNA complex presents a complementary counterpoint to the interactions observed in the Oxytricha nova telomere end-binding and Schizosaccharomyces pombe Pot1 complexes. Analysis of the Cdc13-DBD/ssDNA complex indicates that molecular recognition of extended single-stranded nucleic acids may proceed via a folding-type mechanism rather than resulting from specific patterns of hydrogen bonds. The structure reported here provides a foundation for understanding the mechanism by which Cdc13 recognizes GT-rich heterogeneous sequences with both unusually strong affinity and high specificity.

Systematic analysis of CRISPR-Cas9 mismatch tolerance reveals low levels of off-target activity.

The discovery that the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) acquired immune system can be utilized to create double-strand breaks (DSBs) in eukaryotic genomes has resulted in the ability to create genomic changes more easily than with other genome engineering techniques. While there is significant potential for the CRISPR-Cas9 system to advance basic and applied research, several unknowns remain, including the specificity of the RNA-directed DNA cleavage by the small targeting RNA, the CRISPR RNA (crRNA). Here we describe a novel synthetic RNA approach that allows for high-throughput gene editing experiments. This was used with a functional assay for protein disruption to perform high-throughput analysis of crRNA activity and specificity. We performed a comprehensive test of target cleavage using crRNAs that contain one and two nucleotide mismatches to the DNA target in the 20mer targeting region of the crRNA, allowing for the evaluation of hundreds of potential mismatched target sites without the requirement for the off-target sequences and their adjacent PAMs to be present in the genome. Our results demonstrate that while many crRNAs are functional, less than 5% of crRNAs with two mismatches to their target are effective in gene editing; this suggests an overall high level of functionality but low level of off-targeting.

High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing.

Small RNA cloning and sequencing is uniquely positioned as a genome-wide approach to quantify miRNAs with single-nucleotide resolution. However, significant biases introduced by RNA ligation in current protocols lead to inaccurate miRNA quantification by 1000-fold. Here we report an RNA cloning method that achieves over 95% efficiency for both 5' and 3' ligations. It achieves accurate quantification of synthetic miRNAs with less than two-fold deviation from the anticipated value and over a dynamic range of four orders of magnitude. Taken together, this high-efficiency RNA cloning method permits accurate genome-wide miRNA profiling from total RNAs.

Off-target effects by siRNA can induce toxic phenotype.

Although recent microarray studies have provided evidence of RNA interference (RNAi)-mediated off-target gene modulation, little is known about whether these changes induce observable phenotypic outcomes. Here we show that a fraction of randomly selected small inhibitory RNAs (siRNAs) can induce changes in cell viability in a target-independent fashion. The observed toxicity requires an intact RNAi pathway and can be eliminated by the addition of chemical modifications that reduce off-target effects. Furthermore, an analysis of toxic and nontoxic duplexes identifies a strong correlation between the toxicity and the presence of a 4-base-pair motif (UGGC) in the RISC-entering strand of toxic siRNA. This article provides further evidence of siRNA-induced off-target effects generating a measurable phenotype and also provides an example of how such undesirable phenotypes can be mitigated by addition of chemical modifications to the siRNA.

Induction of the interferon response by siRNA is cell type- and duplex length-dependent.

Long (27-29-bp dsRNA) Dicer-dependent substrates have been identified as potent mediators of RNAi-induced gene knockdown in HEK293 and HeLa cells. As the lengths of these molecules are reported to be below the threshold generally regarded as necessary for induction of the mammalian interferon (IFN) response, these long siRNA are being considered as RNAi substrates in both research and therapeutic settings. In this report, we demonstrate that >23-bp dsRNA can influence cell viability and induce a potent IFN response (highlighted by a strong up-regulation of the dsRNA receptor, Toll-like receptor 3) in a cell type-specific manner. This finding suggests that the length threshold for siRNA induction of the IFN response is not fixed but instead varies significantly among different cell types. Given the diversity of cell types that comprise whole organisms, these findings suggest great care should be taken when considering length variations of dsRNA molecules for RNAi experimentation, especially in therapeutic applications.

3' UTR seed matches, but not overall identity, are associated with RNAi off-targets.

Off-target gene silencing can present a notable challenge in the interpretation of data from large-scale RNA interference (RNAi) screens. We performed a detailed analysis of off-targeted genes identified by expression profiling of human cells transfected with small interfering RNA (siRNA). Contrary to common assumption, analysis of the subsequent off-target gene database showed that overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA, except for near-perfect matches. Instead, off-targeting is associated with the presence of one or more perfect 3' untranslated region (UTR) matches with the hexamer or heptamer seed region (positions 2-7 or 2-8) of the antisense strand of the siRNA. These findings have strong implications for future siRNA design and the application of RNAi in high-throughput screening and therapeutic development.

Site-directed mutagenesis reveals the thermodynamic requirements for single-stranded DNA recognition by the telomere-binding protein Cdc13.

The essential Saccharomyces cerevisiae protein Cdc13 binds the conserved single-stranded overhang at the end of telomeres and mediates access of protein complexes involved in both end-capping and telomerase activity. The single-stranded DNA-binding domain (ssDBD) of Cdc13 exhibits both high affinity (K(d) of 3 pM) and sequence specificity for the GT-rich sequences present at yeast telomeres. We have used the ssDBD of Cdc13 to understand the sequence-specific recognition of extended single-stranded DNA (ssDNA). The recent structure of the Cdc13 DNA-binding domain revealed that ssDNA is recognized by a large protein surface containing an oligonucleotide/oligosaccharide-binding fold (OB-fold) augmented by an extended 30-amino acid loop. Contacts to ssDNA occur via a contiguous surface of aromatic, hydrophobic, and basic residues. A complete alanine scan of the binding interface has been used to determine the contribution of each contacting side chain to binding affinity. Substitution of any aromatic or hydrophobic residue at the interface was deleterious to binding (20 to >700-fold decrease in binding affinity), while tolerance for replacement of basic residues was observed. The important aromatic and hydrophobic contacts are spread throughout the extended interface, indicating that the entire surface is both structurally and thermodynamically required for binding. While all of these contacts are important, several of the individual alanine substitutions that abolish binding cluster to one region of the protein surface. This region is vital for recognition of four bases at the 5' end of the DNA and constitutes a "hotspot" of binding affinity.

Conserved structure for single-stranded telomeric DNA recognition.

The essential Cdc13 protein in the yeast Saccharomyces cerevisiae is a single-stranded telomeric DNA binding protein required for chromosome end protection and telomere replication. Here we report the solution structure of the Cdc13 DNA binding domain in complex with telomeric DNA. The structure reveals the use of a single OB (oligonucleotide/oligosaccharide binding) fold augmented by an unusually large loop for DNA recognition. This OB fold is structurally similar to OB folds found in the ciliated protozoan telomere end-binding protein, although no sequence similarity is apparent between them. The common usage of an OB fold for telomeric DNA interaction demonstrates conservation of end-protection mechanisms among eukaryotes.

The poxvirus p28 virulence factor is an E3 ubiquitin ligase.

A majority of the orthopoxviruses, including the variola virus that causes the dreaded smallpox disease, encode a highly conserved 28-kDa protein with a classic RING finger sequence motif (C(3)HC(4)) at their carboxyl-terminal domains. The RING domain of p28 has been shown to be a critical determinant of viral virulence for the ectromelia virus (mousepox virus) in a murine infection model (Senkevich, T. G., Koonin, E. V., and Buller, R. M. (1994) Virology 198, 118-128). Here, we demonstrate that the p28 proteins encoded by the ectromelia virus and the variola virus possess E3 ubiquitin ligase activity in biochemical assays as well as in cultured mammalian cells. Point mutations disrupting the RING finger domain of p28 completely abolish its E3 ligase activity. In addition, p28 functions cooperatively with Ubc4 and UbcH5c, the E2 conjugating enzymes involved in 26 S proteasome degradation of protein targets. Moreover, p28 catalyzes the formation of Lys-63-linked polyubiquitin chains in the presence of Ubc13/Uev1A, a heterodimeric E2 conjugating enzyme, indicating that p28 may regulate the biological activity of its cognate viral and/or host cell target(s) by Lys-63-linked ubiquitin multimers. We thus conclude that the poxvirus p28 virulence factor is a new member of the RING finger E3 ubiquitin ligase family and has a unique polyubiquitylation activity. We propose that the E3 ligase activity of the p28 virulence factor may be targeted for therapeutic intervention against infections by the variola virus and other poxviruses.