Methods in molecular biology
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With rapid advances in experimental instruments and protocols, imaging and sequencing data are being generated at an unprecedented rate contributing significantly to the current and coming big biomedical data. Meanwhile, unprecedented advances in computational infrastructure and analysis algorithms are realizing image-based digital diagnosis not only in radiology and cardiology but also oncology and other diseases. Machine learning methods, especially deep learning techniques, are already and broadly implemented in diverse technological and industrial sectors, but their applications in healthcare are just starting. ⋯ Moreover, the applications of genomics data are realizing the potential for personalized medicine, making diagnosis, treatment, monitoring, and prognosis more accurate. In this chapter, we discuss machine learning methods readily available for digital pathology applications, new prospects of integrating spatial genomics data on tissues with tissue morphology, and frontier approaches to combining genomics data with pathological imaging data. We present perspectives on how artificial intelligence can be synergized with molecular genomics and imaging to make breakthroughs in biomedical and translational research for computer-aided applications.
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The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system, now reclassified as Cas12a, is a DNA-editing platform analogous to the widely used CRISPR-Cas9 system. The Cas12a system exhibits several distinct features over the CRISPR-Cas9 system, such as increased specificity and a smaller gene size to encode the nuclease and the matching CRISPR guide RNA (crRNA), which could mitigate off-target and delivery problems, respectively, described for the Cas9 system. However, the Cas12a system exhibits reduced gene editing efficiency compared to Cas9. ⋯ To optimize the CRISPR-Cas12a system, we describe the inclusion of a self-cleaving ribozyme in the vector design to facilitate accurate 3'-end processing of the crRNA transcript to produce precise molecules. This optimized design enhanced not only the gene editing efficiency, but also the activity of the catalytically inactive Cas12a-based CRISPR gene activation platform. We thus generated an improved CRISPR-Cas12a system for more efficient gene editing and gene regulation purposes.
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The number of studies published in the biomedical literature has dramatically increased over the last few decades. This massive proliferation of literature makes clinical medicine increasingly complex, and information from multiple studies is often needed to inform a particular clinical decision. However, available studies often vary in their design, methodological quality, and population studied, and may define the research question of interest quite differently. ⋯ In addition, since even highly cited trials may be challenged over time, clinical decision-making requires ongoing reconciliation of studies which provide different answers to the same question. Because it is often impractical for readers to track down and review all the primary studies, systematic reviews and meta-analyses are an important source of evidence on the diagnosis, prognosis and treatment of any given disease. This chapter summarizes methods for conducting and reading systematic reviews and meta-analyses, as well as describes potential advantages and disadvantages of these publications.
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The intention-to-treat analysis is the gold standard for evaluating the efficacy in a randomized controlled trial. However, when non-adherence to randomized treatments is high the actual treatment effect may be underestimated. ⋯ These analyses may include censoring at the time of co-interventions associated with stopping treatment, lag censoring which allows an additional period after discontinuation of study treatment to account for residual treatment effects, inverse probability of censoring weights (IPCW), accelerated failure time models, and contamination adjusted intent-to-treat analysis. These methods are particularly useful in assessing the "prescribed efficacy" of the study treatment, which can aid clinical decision-making .
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CRISPR-Cas9 gene editing is dependent on a programmable single guide RNA (sgRNA) that directs Cas9 endonuclease activity. This RNA is often generated by enzymatic reactions, however the process becomes time-consuming as the number of sgRNAs increases and does not allow the incorporation of chemical modifications that can improve or expand the functionality of CRISPR. ⋯ Here, we demonstrate a "split-and-click" approach that separates the sgRNA into its two smaller components - a DNA-targeting ~20-mer RNA and a constant Cas9-binding 79-mer RNA - and chemically ligates them together to generate a biologically active sgRNA. The benefits of our approach lie in the stringent purification of the DNA-targeting 20-mer, the reduced synthesis of the constant 79-mer each time a new sgRNA is required, and the rapid access it provides to custom libraries of sgRNAs.