Novel and Engineered Type II CRISPR Systems from Uncultivated Microbes with Broad Genome Editing Capability.

Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes. We developed a high-throughput pipeline that enabled us to predict tracrRNA sequences, to design single guide RNAs, and to demonstrate nuclease activity in vitro for 41 newly described subgroups. Active systems represent an extensive diversity of protein sequences and guide RNA structures and require diverse protospacer adjacent motifs (PAMs) that collectively expand the known targeting capability of current systems. Several nucleases showed activity levels comparable to or significantly higher than SpCas9, despite being smaller in size. In addition, top systems exhibited low levels of off-target editing in mammalian cells, and PAM-interacting domain engineered chimeras further expanded their targetability. These newly discovered nucleases are attractive enzymes for translation into many applications, including therapeutics.

Compact Cas9d and HEARO enzymes for genome editing discovered from uncultivated microbes.

Programmable, RNA-guided nucleases are diverse enzymes that have been repurposed for biotechnological applications. However, to further expand the therapeutic application of these tools there is a need for targetable systems that are small enough to be delivered efficiently. Here, we mined an extensive genome-resolved metagenomics database and identified families of uncharacterized RNA-guided, compact nucleases (between 450 and 1,050 aa). We report that Cas9d, a new CRISPR type II subtype, contains Zinc-finger motifs and high arginine content, features that we also found in nucleases related to HEARO effectors. These enzymes exhibit diverse biochemical characteristics and are broadly targetable. We show that natural Cas9d enzymes are capable of genome editing in mammalian cells with >90% efficiency, and further engineered nickase variants into the smallest base editors active in E. coli and human cells. Their small size, broad targeting potential, and translatability suggest that Cas9d and HEARO systems will enable a variety of genome editing applications.

Novel Type V-A CRISPR Effectors Are Active Nucleases with Expanded Targeting Capabilities.

Cas12a enzymes are quickly being adopted for use in a variety of genome-editing applications. These programmable nucleases are part of adaptive microbial immune systems, the natural diversity of which has been largely unexplored. Here, we identified novel families of Type V-A CRISPR nucleases through a large-scale analysis of metagenomes collected from a variety of complex environments, and developed representatives of these systems into gene-editing platforms. The nucleases display extensive protein variation and can be programmed by a single-guide RNA with specific motifs. The majority of these enzymes are part of systems recovered from uncultivated organisms, some of which also encode a divergent Type V effector. Biochemical analysis uncovered unexpected protospacer adjacent motif diversity, indicating that these systems will facilitate a variety of genome-engineering applications. The simplicity of guide sequences and activity in human cell lines suggest utility in gene and cell therapies.

Non-equilibrium dynamics of a nascent polypeptide during translation suppress its misfolding.

Protein folding can begin co-translationally. Due to the difference in timescale between folding and synthesis, co-translational folding is thought to occur at equilibrium for fast-folding domains. In this scenario, the folding kinetics of stalled ribosome-bound nascent chains should match the folding of nascent chains in real time. To test if this assumption is true, we compare the folding of a ribosome-bound, multi-domain calcium-binding protein stalled at different points in translation with the nascent chain as is it being synthesized in real-time, via optical tweezers. On stalled ribosomes, a misfolded state forms rapidly (1.5 s). However, during translation, this state is only attained after a long delay (63 s), indicating that, unexpectedly, the growing polypeptide is not equilibrated with its ensemble of accessible conformations. Slow equilibration on the ribosome can delay premature folding until adequate sequence is available and/or allow time for chaperone binding, thus promoting productive folding.

Validating functional measures of physical ability for aging people with intellectual developmental disability.

Unlike the aging population without intellectual and developmental disabilities (IDD), few standardized performance measures exist to assess physical function and risk for adverse outcomes such as nonfatal, unintentional injuries. We modified 3 selected standardized performance tools in the areas of general fitness (2-Minute Walk Test), balance and gait (Performance-Oriented Mobility Assessment I), and functional independence (Modified Barthel Index) for administration with people with IDD. The modified tools were piloted with 30 participants. Results indicated the measures are strongly associated and successfully distinguished between participants with an adverse health event in the previous year. The modified tools have potential to provide clinicians with quantitative measures that track physical performance changes associated with aging in people with IDD.

Patient navigation significantly reduces delays in breast cancer diagnosis in the District of Columbia.

BACKGROUND: Patient Navigation (PN) originated in Harlem as an intervention to help poor women overcome access barriers to timely breast cancer treatment. Despite rapid, nationally widespread adoption of PN, empirical evidence on its effectiveness is lacking. In 2005, National Cancer Institute initiated a multicenter PN Research Program (PNRP) to measure PN effectiveness for several cancers. The George Washington Cancer Institute, a project participant, established District of Columbia (DC)-PNRP to determine PN's ability to reduce breast cancer diagnostic time (number of days from abnormal screening to definitive diagnosis). METHODS: A total of 2,601 women (1,047 navigated; 1,554 concurrent records-based nonnavigated) were examined for breast cancer from 2006 to 2010 at 9 hospitals/clinics in DC. Analyses included only women who reached complete diagnostic resolution. Differences in diagnostic time between navigation groups were tested with ANOVA models including categorical demographic and treatment variables. Log transformations normalized diagnostic time. Geometric means were estimated and compared using Tukey-Kramer P value adjustments. RESULTS: Average-geometric mean [95% confidence interval (CI)]-diagnostic time (days) was significantly shorter for navigated, 25.1 (21.7, 29.0), than nonnavigated women, 42.1 (35.8, 49.6). Subanalyses revealed significantly shorter average diagnostic time for biopsied navigated women, 26.6 (21.8, 32.5) than biopsied nonnavigated women, 57.5 (46.3, 71.5). Among nonbiopsied women, diagnostic time was shorter for navigated, 27.2 (22.8, 32.4), than nonnavigated women, 34.9 (29.2, 41.7), but not statistically significant. CONCLUSIONS: Navigated women, especially those requiring biopsy, reached their diagnostic resolution significantly faster than nonnavigated women. IMPACT: Results support previous findings of PN's positive influence on health care. PN should be a reimbursable expense to assure continuation of PN programs.

Having health insurance does not eliminate race/ethnicity-associated delays in breast cancer diagnosis in the District of Columbia.

BACKGROUND: Delays in follow-up after breast cancer screening contribute to disparities in breast cancer outcomes. The objective of this research was to determine the impact of race/ethnicity and health insurance on diagnostic time, defined as number of days from suspicious finding to diagnostic resolution. METHODS: This retrospective cohort study of 1538 women examined for breast abnormalities between 1998-2010 at 6 hospitals/clinics in the District of Columbia measured mean diagnostic times between non-Hispanic whites (NHWs), non-Hispanic blacks (NHBs), and Hispanics with private, government, or no health insurance by using a full-factorial ANOVA model. RESULTS: Respective average--geometric mean (95% CI)--diagnostic times (in days) for NHWs, NHBs, and Hispanics were 16 (12, 21), 27 (23, 33), and 51 (35, 76) among privately insured; 12 (7, 19), 39 (32, 48), and 71 (48, 105) among government insured; 45 (17, 120), 60 (39, 92), and 67 (56, 79) among uninsured. Government insured NHWs had significantly shorter diagnostic times than government insured NHBs (P = .0003) and Hispanics (P < .0001). Privately insured NHWs had significantly shorter diagnostic times than privately insured NHBs (P = .03) and Hispanics (P < .0001). Privately insured NHBs had significantly shorter diagnostic times than uninsured NHBs (P = .03). CONCLUSIONS: Insured minorities waited >2 times longer to reach their diagnostic resolution than insured NHWs. Having private health insurance increased the speed of diagnostic resolution in NHBs; however, their diagnostic time remained significantly longer than for privately insured NHWs. These results suggest diagnostic delays in minorities are more likely caused by other barriers associated with race/ethnicity than by insurance status.

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.