Temporal restriction of Cas9 expression improves CRISPR-mediated deletion efficacy and fidelity.

Clinical application of CRISPR-Cas9 technology for large deletions of somatic mutations is inefficient, and methods to improve utility suffer from our inability to rapidly assess mono- vs. biallelic deletions. Here we establish a model system for investigating allelic heterogeneity at the single-cell level and identify indel scarring from non-simultaneous nuclease activity at gRNA cut sites as a major barrier to CRISPR-del efficacy both in vitro and in vivo. We show that non-simultaneous nuclease activity is partially prevented via restriction of CRISPR-Cas9 expression via inducible adeno-associated viruses (AAVs) or lipid nanoparticles (LNPs). Inducible AAV-based expression of CRISPR-del machinery significantly improved mono- and biallelic deletion frequency in vivo, supporting the use of the Xon cassette over traditional constitutively expressing AAV approaches. These data depicting improvements to deletions and insight into allelic heterogeneity after CRISPR-del will inform therapeutic approaches for phenotypes that require either large mono- or biallelic deletions, such as autosomal recessive diseases or where mutant allele-specific gRNAs are not readily available, or in situations where the targeted sequence for excision is located multiple times in a genome.

Standard screening methods underreport AAV-mediated transduction and gene editing.

Conventional methods to discern adeno-associated virus (AAV) vector transduction patterns are based on high, stable expression of a reporter gene. As a consequence, conventionally described tropisms omit cell types that undergo transient transduction, or have low but undetectable levels of reporter expression. This creates a blind spot for AAV-based genome editing applications because only minimal transgene expression is required for activity. Here, we use editing-reporter mice to fill this void. Our approach sensitively captures both high and low transgene expression from AAV vectors. Using AAV8 and other serotypes, we demonstrate the superiority of the approach in a side-by-side comparison with traditional methods, demonstrate numerous, previously unknown sites of AAV targeting, and better predict the gene editing footprint after AAV-CRISPR delivery. We anticipate that this system, which captures the full spectrum of transduction patterns from AAV vectors in vivo, will be foundational to current and emerging AAV technologies.

Kinetic Trapping of Folded Proteins Relative to Aggregates under Physiologically Relevant Conditions.

Anfinsen's thermodynamic hypothesis does not explicitly take into account the possibility of protein aggregation. Here, we introduce a cyclic-perturbation approach to prove that not only the native state but also soluble aggregates of most proteins can be highly populated under mild, physiologically relevant conditions, even at very low concentration. Surprisingly, these aggregates are not necessarily amyloid in nature and are usually not observed in bioactive proteins due to the extremely low kinetic flux from the native state toward a region of the chemical-potential landscape encoding aggregates. We first illustrate this concept for the representative model protein apomyoglobin-at room temperature and no denaturant-and demonstrate kinetic trapping of the native state relative to at least two different types of soluble, predominantly nonamyloid aggregates. The concentration and temperature dependence of aggregation confirm the above scenario. Extension of our analysis to the Escherichia coli proteome shows that the majority of the soluble bacterial proteome is also kinetically trapped in the nonaggregated state. Hence, the existence and low kinetic accessibility of large aggregates at room temperature and pH 6-7 is a general phenomenon. We also show that the average critical protein concentration for aggregation of most of the bacterial proteome is extremely small, much lower than the typical cellular protein concentration. Hence, the thermodynamic driving force for protein aggregation is large even if aggregation does not usually occur in healthy cells due to kinetic trapping. A broader view of Anfinsen's thermodynamic hypothesis encompassing all protein states, including aggregates, is necessary to understand the behavior of proteins in their natural environment.

Johan Turi's animal, mineral, vegetable cures and healing practices: an in-depth analysis of Sami (Saami) folk healing one hundred years ago.

BACKGROUND: The healing knowledge of a Sami (Saami) hunter and reindeer herder was surveyed as a window into the concepts of health, healing, and disease in early twentieth-century Sapmi (Northern Sweden). The two books of Johan Turi (1854-1936)--An Account of the Sami (1910) and Lappish Texts (1918-19) were examined to determine the varieties of recorded zootherapeutic, mineral, chemical, and ethnobotanical lore, as well as the therapeutic acts, identified conditions, and veterinary knowledge included. METHODS: Tabulation of the materials and species mentioned in Turi's descriptions (n = 137) permitted analysis of the relative frequency of differing types of healing in Turi's overall therapeutic repertoire, his relative attention to chronic vs. acute ailments, and the frequency of magic as a component of healing. A qualitative appraisal was made of the degree to which outside influences affected Sami healing of the period. A further assessment of the possible clinical efficacy of the recorded remedies was undertaken. RESULTS: Turi's remedies consist most often of zootherapeutics (31%), followed by physical acts such as massage, moxibustion, or manipulation (22%). Ethnobotanical cures make up a significantly smaller portion of his repertoire (17%), followed by mineral and chemical cures (12%). Magic rituals (including incantations and ritual acts) make up a significant portion of Turi's repertoire, and could be used alone (17%) or in conjunction with other types of healing (38%). Turi's healing aimed primarily at acute ailments (65%), with chronic conditions addressed less often (35%). A literature review revealed that Turi's remedies held a marked frequency of likely efficacy, at least in cases in which it was possible to ascertain the precise species, conditions, or substances described. Although it is possible at times to recognize foreign sources in Turi's repertoire, it is clear that Turi understood all his healing methods as distinctively Sami. CONCLUSION: The research illustrates the variety and depth of a single informant's healing knowledge, and demonstrates the value of both historical sources and in-depth data collection with single experts as useful means of assessing and characterizing an indigenous population's healing traditions.