Targeted nonviral delivery of genome editors in vivo.

Cell-type-specific in vivo delivery of genome editing molecules is the next breakthrough that will drive biological discovery and transform the field of cell and gene therapy. Here, we discuss recent advances in the delivery of CRISPR-Cas genome editors either as preassembled ribonucleoproteins or encoded in mRNA. Both strategies avoid pitfalls of viral vector-mediated delivery and offer advantages including transient editor lifetime and potentially streamlined manufacturing capability that are already proving valuable for clinical use. We review current applications and future opportunities of these emerging delivery approaches that could make genome editing more efficacious and accessible in the future.

CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

A mitochondria-specific mutational signature of aging: increased rate of A > G substitutions on the heavy strand.

The mutational spectrum of the mitochondrial DNA (mtDNA) does not resemble any of the known mutational signatures of the nuclear genome and variation in mtDNA mutational spectra between different organisms is still incomprehensible. Since mitochondria are responsible for aerobic respiration, it is expected that mtDNA mutational spectrum is affected by oxidative damage. Assuming that oxidative damage increases with age, we analyse mtDNA mutagenesis of different species in regards to their generation length. Analysing, (i) dozens of thousands of somatic mtDNA mutations in samples of different ages (ii) 70053 polymorphic synonymous mtDNA substitutions reconstructed in 424 mammalian species with different generation lengths and (iii) synonymous nucleotide content of 650 complete mitochondrial genomes of mammalian species we observed that the frequency of AH > GH substitutions (H: heavy strand notation) is twice bigger in species with high versus low generation length making their mtDNA more AH poor and GH rich. Considering that AH > GH substitutions are also sensitive to the time spent single-stranded (TSSS) during asynchronous mtDNA replication we demonstrated that AH > GH substitution rate is a function of both species-specific generation length and position-specific TSSS. We propose that AH > GH is a mitochondria-specific signature of oxidative damage associated with both aging and TSSS.

Methylation-Sensitive Restriction Enzyme Quantitative Polymerase Chain Reaction Enables Rapid, Accurate, and Precise Detection of Methylation Status of the Regulatory T Cell (Treg)-Specific Demethylation Region in Primary Human Tregs.

Human regulatory T cells (Tregs) have been implicated in cancer immunotherapy and are also an emerging cellular therapeutic for the treatment of multiple indications. Although Treg stability during ex vivo culture has improved, methods to assess Treg stability such as bisulfite Sanger sequencing to determine the methylation status of the Treg-specific demethylated region (TSDR) have remained unchanged. Bisulfite Sanger sequencing is not only costly and cumbersome to perform, it is inaccurate because of relatively low read counts. Bisulfite next-generation sequencing, although more accurate, is a less accessible method. In this study, we describe the application of methylation-sensitive restriction enzymes (MSRE) and quantitative PCR (qPCR) to determine the methylation status of the TSDR. Using known ratios of Tregs and non-Tregs, we show that MSRE-qPCR can distinguish the methylation status of the TSDR in populations of cells containing increasing proportions of Tregs from 0 to 100%. In a comparison with values obtained from an established bisulfite next-generation sequencing approach for determining the methylation status of the TSDR, our MSRE-qPCR results were within 5% on average for all samples with a high percentage (>70%) of Tregs, reinforcing that MSRE-qPCR can be completed in less time than other methods with the same level of accuracy. The value of this assay was further demonstrated by quantifying differences in TSDR methylation status of Tregs treated with and without rapamycin during an ex vivo expansion culture. Together, we show that our novel application of the MSRE-qPCR to the TSDR is an optimal assay for accurate assessment of Treg purity.

AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines.

Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, "AsCas12a Ultra", that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

Medical practice in rural Saskatchewan: factors in physician recruitment and retention.

INTRODUCTION: The recruitment and retention of physicians in rural communities is a challenge throughout Canada and across the globe. In 1976, a group of medical students profiled rural communities with medical practices and produced a summary report entitled Medical Practice in Saskatchewan. Our objective was to repeat the 1976 study and to identify factors that motivate physicians to select rural locations for practice. METHODS: Physicians practising in rural Saskatchewan were interviewed in 2011 and 2012. Through qualitative, inductive analysis, we identified themes that drove the recruitment and retention of physicians. RESULTS: Sixty-two physicians were interviewed and 105 communities profiled. Of the physicians interviewed, 21 noted that the ability to practise full-scope family medicine and having the freedom to practise as they desire was important for recruitment, and 43 reported that these factors influenced their decision to remain in a community. Attraction to a rural lifestyle (cited by 17 physicians), having a rural background (13) and having ties to a specific community (12) were important for recruitment. Feeling appreciated by patients (45), one's spouse and/or family enjoying the community (41), and integration into the community (38) were important factors for retention. CONCLUSION: The decision to practise in a rural location correlates with a desire for a broad and varied scope of practice, being attracted to a rural lifestyle and having rural roots. Once physicians establish a rural practice, they are more likely to stay if they can continue a broad scope of practice, if they feel appreciated by their patients, and if their spouses and family are happy in the community.

INTRODUCTION: Le recrutement et la fidélisation des médecins dans les communautés rurales présentent un défi partout au Canada et ailleurs dans le monde. En 1976, un groupe d'étudiants en médecine a dressé un profil des communautés rurales dotées de pratiques médicales et produit un rapport sommaire intitulé Medical Practice in Saskatchewan. Notre objectif est de répéter l'étude de 1976 et de relever les facteurs qui motivent les médecins à choisir de pratiquer en région rurale. MÉTHODES: Nous avons interrogé des médecins exerçant en milieu rural en Saskatchewan en 2011 et en 2012. Par analyse inductive qualitative, nous avons recensé les facteurs qui ont favorisé leur recrutement et leur fidélisation. RÉSULTATS: Soixante-deux médecins ont été interrogés et le profil de 105 communautés a été dressé. Parmi les médecins interrogés, 21 ont mentionné que la capacité d'exercer pleinement dans toute l'envergure de la médecine familiale et la liberté de pratiquer comme ils le souhaitaient avaient joué un rôle important dans leur recrutement et 43 ont mentionné que ces facteurs avaient influé sur leur décision de demeurer dans une communauté. Les facteurs suivants ont aussi été jugés importants pour le recrutement : l'attrait du mode de vie rural (mentionné par 17 médecins), provenir d'un milieu rural (13) et les liens avec une communauté en particulier (12). En ce qui concerne les facteurs de fidélisation, le sentiment d'être apprécié par les patients (45), le fait que les conjoints et(ou) les proches apprécient la vie dans la communauté (41) et l'intégration dans la communauté (38). CONCLUSION: La décision d'exercer en milieu rural est en corrélation avec le désir d'avoir une pratique dont l'envergure est vaste et variée, l'attirance pour un mode de vie rural et des racines rurales. Une fois que les médecins s'établissent en milieu rural, ils sont plus susceptibles d'y rester s'ils peuvent continuer d'avoir une pratique diversifiée, s'ils se sentent appréciés par leurs patients et si les conjoints et les familles se plaisent dans la communauté.