RESUMO
Detailed target-selectivity information and experiment-based efficacy prediction tools are primarily available for Streptococcus pyogenes Cas9 (SpCas9). One obstacle to develop such tools is the rarity of accurate data. Here, we report a method termed 'Self-targeting sgRNA Library Screen' (SLS) for assaying the activity of Cas9 nucleases in bacteria using random target/sgRNA libraries of self-targeting sgRNAs. Exploiting more than a million different sequences, we demonstrate the use of the method with the SpCas9-HF1 variant to analyse its activity and reveal motifs that influence its target-selectivity. We have also developed an algorithm for predicting the activity of SpCas9-HF1 with an accuracy matching those of existing tools. SLS is a facile alternative to the much more expensive and laborious approaches used currently and has the capability of delivering sufficient amount of data for most of the orthologs and variants of SpCas9.
Assuntos
Proteína 9 Associada à CRISPR , RNA/química , Algoritmos , Animais , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Linhagem Celular Tumoral , Clivagem do DNA , Variação Genética , Camundongos , Streptococcus pyogenes/enzimologiaRESUMO
Streptococcus pyogenes Cas9 (SpCas9) has been employed as a genome engineering tool with a promising potential within therapeutics. However, its off-target effects present major safety concerns for applications requiring high specificity. Approaches developed to date to mitigate this effect, including any of the increased-fidelity (i.e., high-fidelity) SpCas9 variants, only provide efficient editing on a relatively small fraction of targets without detectable off-targets. Upon addressing this problem, we reveal a rather unexpected cleavability ranking of target sequences, and a cleavage rule that governs the on-target and off-target cleavage of increased-fidelity SpCas9 variants but not that of SpCas9-NG or xCas9. According to this rule, for each target, an optimal variant with matching fidelity must be identified for efficient cleavage without detectable off-target effects. Based on this insight, we develop here an extended set of variants, the CRISPRecise set, with increased fidelity spanning across a wide range, with differences in fidelity small enough to comprise an optimal variant for each target, regardless of its cleavability ranking. We demonstrate efficient editing with maximum specificity even on those targets that have not been possible in previous studies.
Assuntos
Engenharia , Streptococcus pyogenes , Streptococcus pyogenes/genéticaRESUMO
INTRODUCTION: Squamous cell carcinomas (SCC) are a major subgroup of malignant tumors with a platinum-based first-line systematic chemotherapy. miRNAs play a role in various diseases and modulate therapy response as well. The aim of this study was to identify predictive miRNAs in platinum-treated SCCs. METHODS: miRNA expression data of platinum-treated head and neck (HNSC), cervical (CESC) and lung (LUSC) cancer were collected from the TCGA repositories. Treatment response was defined based on presence or absence of disease progression at 18 months. Responder and nonresponder cohorts were compared using Mann-Whitney and Receiver Operating Characteristic tests. Logistic regression was developed to establish a predictive miRNA signature. Significance was set at FDR < 5%. RESULTS: The integrated database includes 266 SCC patient samples with platinum-based therapy and available follow-up. We uncovered 16, 103, and 9 miRNAs correlated to chemotherapy response in the CESC, HNSC, and LUSC cohorts, respectively. Eight miRNAs overlapped between the CESC and HNSC subgroups, and three miRNAs overlapped between the LUSC and HNSC subgroups. We established a logistic regression model in HNSC and CESC which included six miRNAs: hsa-miR-5586 (Exp (B): 2.94, p = 0.001), hsa-miR-632 (Exp (B): 10.75, p = 0.002), hsa-miR-2355 (Exp (B): 0.48, p = 0.004), hsa-miR-642a (Exp (B): 2.22, p = 0.01), hsa-miR-101-2 (Exp (B): 0.39, p = 0.013) and hsa-miR-6728 (Exp (B): 0.21, p = 0.016). The model using these miRNAs was able to predict chemotherapy resistance with an AUC of 0.897. CONCLUSIONS: We performed an analysis of RNA-seq data of squamous cell carcinomas samples and identified significant miRNAs correlated to the response against platinum-based therapy in cervical, head and neck, and lung tumors.