Sniper2L is a high-fidelity Cas9 variant with high activity.

Although several high-fidelity SpCas9 variants have been reported, it has been observed that this increased specificity is associated with reduced on-target activity, limiting the applications of the high-fidelity variants when efficient genome editing is required. Here, we developed an improved version of Sniper-Cas9, Sniper2L, which represents an exception to this trade-off trend as it showed higher specificity with retained high activity. We evaluated Sniper2L activities at a large number of target sequences and developed DeepSniper, a deep learning model that can predict the activity of Sniper2L. We also confirmed that Sniper2L can induce highly efficient and specific editing at a large number of target sequences when it is delivered as a ribonucleoprotein complex. Mechanically, the high specificity of Sniper2L originates from its superior ability to avoid unwinding a target DNA containing even a single mismatch. We envision that Sniper2L will be useful when efficient and specific genome editing is required.

Single Molecule FRET Analysis of CRISPR Cas9 Single Guide RNA Folding Dynamics.

CRISPR Cas9 is an RNA guided endonuclease that is part of a bacterial adaptive immune system. Single guide RNA (sgRNA) can be designed to target genomic DNA, making Cas9 a programmable DNA binding/cutting enzyme and allowing applications such as epigenome editing, controlling transcription, and targeted DNA insertion. Some of the main hurdles against an even wider adoption are off-target effects and variability in Cas9 editing outcomes. Most studies that aim to understand the mechanisms that underlie these two areas have focused on Cas9 DNA binding, DNA unwinding, and target cleavage. The assembly of Cas9 RNA ribonucleoprotein complex (RNP) precedes all these steps and includes sgRNA folding and Cas9 binding to sgRNA. We know from the crystal structure of the Cas9 RNP what the final sgRNA conformation is. However, the assembly dynamics has not been studied in detail and a better understanding of RNP assembly could lead to better-designed sgRNAs and better editing outcomes. To study this process, we developed a single molecule FRET assay to monitor the conformation of the sgRNA and the binding of Cas9 to sgRNA. We labeled the sgRNA with a donor fluorophore and an acceptor fluorophore such that when the sgRNA folds, there are changes in FRET efficiency. We measured sgRNA folding dynamics under different ion conditions, under various methods of folding (refolding vs vectorial), and with or without Cas9. sgRNA that closely mimics the sgRNA construct used for high resolution structural analysis of the Cas9-gRNA complex showed two main FRET states without Cas9, and Cas9 addition shifted the distribution toward the higher FRET state attributed to the properly assembled complex. Even in the absence of Cas9, folding the sgRNA vectorially using a superhelicase-dependent release of the sgRNA in the direction of transcription resulted in almost exclusively high FRET state. An addition of Cas9 during vectorial folding greatly reduced a slow-folding fraction. Our studies shed light on the heterogeneous folding dynamics of sgRNA and the impact of co-transcriptional folding and Cas9 binding in sgRNA folding. Further studies of sequence dependence may inform rational design of sgRNAs for optimal function.

Single molecule methods for studying CRISPR Cas9-induced DNA unwinding.

Like helicases, CRISPR proteins such as Cas9 and Cas12a unwind DNA, but unlike helicases, these CRISPR proteins do not use ATP. Instead, they use binding energy to melt DNA locally and then utilize basepairing between guide (g) RNA and target strand to continue to unwind the DNA. CRISPR Cas9 is the most widely used tool for genome editing applications. The Cas9 endonuclease forms a complex with gRNA that can be programmed to bind a specific 20 bp segment of DNA, the protospacer. If there is enough of a sequence match between sgRNA and protospacer, Cas9 undergoes a conformational change, which activates the two nuclease domains, causing a double strand break in the DNA. We can use single-molecule FRET (smFRET) to probe the state of DNA unwinding as a function of mismatches between sgRNA and DNA. This approach can also be used to probe the position of Cas9's HNH domain before and after cleavage.

Indwelling medical device use and sepsis risk at a health professional shortage area hospital: Possible interaction with length of hospitalization.

BACKGROUND: We aimed to identify risk factors for sepsis diagnosis and possible interaction with length of hospital stay (LOS) among inpatients at a rural Health Professional Shortage Area hospital. METHODS: This case-control study examined 600 adult patients (300 cases and 300 controls) admitted to a rural health system in North Carolina between 2012 and 2018. Case selection was based on assignment of ICD-9-CM diagnostic codes for sepsis. Controls were patients with a medical diagnosis other than sepsis during the observational period. Logistic regression was used to model sepsis diagnosis as a function of indwelling medical device use and stratified by LOS. RESULTS: Indwelling medical device use preadmission and postadmission were significantly associated with increased risk of sepsis diagnosis among patients with extended hospital stays (LOS ≥ 5 days) (odds ratio [OR] = 5.51; 95% confidence interval [CI] = 1.95-15.62; P = .001 and OR = 3.28; 95% CI = 1.24-8.68; P = .017, respectively). Among patients with LOS <5 days, association with sepsis diagnosis was only significant for indwelling medical device use preadmission (OR = 9.61; 95% CI = 3.68-25.08; P < .0001). CONCLUSIONS: Indwelling medical device use was significantly associated with increased risk of sepsis diagnosis and the risk was higher with longer hospitalization.

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding.

Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5' end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5' end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

Cellular and viral oncogenes: the key to unlocking unknowns of Kaposi's sarcoma-associated herpesvirus pathogenesis.

Oncogenic viruses carry an extensive arsenal of oncogenes for hijacking cellular pathways. Notably, variations in oncogenes among tumor-producing viruses give rise to different mechanisms for cellular transformation. Specifically, Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus able to infect and transform a variety of cell types. The oncogenicity of KSHV disseminates from the virus' ability to induce and encode a wide variety of both cellular and viral oncogenes. Such an array of cellular and viral oncogenes enables KSHV to induce the malignant phenotype of a KSHV-associated cancer. Evolutionarily, KSHV has acquired many oncogenic homologues capable of inducing cell proliferation, cell differentiation, cell survival, and immune evasion. Integration between inducing and encoding oncogenes plays a vital role in KSHV pathogenicity. KSHV is alleged to harbor the highest number of potential oncogenes by which a virus promotes cellular transformation and malignancy. Many KSHV inducing/encoding oncogenes are mainly expressed during the latent phase of KSHV infection, a period required for virus establishment of malignant cellular transformation. Elucidation of the exact mechanism(s) by which oncogenes promote KSHV pathogenicity would not only give rise to potential novel therapeutic targets/drugs but would also add to our understanding of cancer biology. The scope of this review is to examine the roles of the most important cellular and viral oncogenes involved in KSHV pathogenicity.