A Guide to Understanding "State-of-the-Art" Basic Research Techniques in Anesthesiology

Abstract

Perioperative medicine is changing from a "protocol-based" approach to a progressively personalized care model. New molecular techniques and comprehensive perioperative medical records allow for detection of patient-specific phenotypes that may better explain, or even predict, a patient's response to perioperative stress and anesthetic care. Basic science technology has significantly evolved in recent years with the advent of powerful approaches that have translational relevance. It is incumbent on us as a primarily clinical specialty to have an in-depth understanding of rapidly evolving underlying basic science techniques to incorporate such approaches into our own research, critically interpret the literature, and improve future anesthesia patient care. This review focuses on 3 important and most likely practice-changing basic science techniques: next-generation sequencing (NGS), clustered regularly interspaced short palindromic repeat (CRISPR) modulations, and inducible pluripotent stem cells (iPSCs). Each technique will be described, potential advantages and limitations discussed, open questions and challenges addressed, and future developments outlined. We hope to provide insight for practicing physicians when confronted with basic science articles and encourage investigators to apply "state-of-the-art" technology to their future experiments.

Figure 1.. Next-generation sequencing (NGS) overview.

1) Preparation of Samples: Multiple different types of samples can be processed to obtain material for NGS. RNA is extracted from bulk, laser-capture microdissected (LCM) or fluorescence-activated cell sorted (FACS) tissues. 2) Library construction: RNA is reverse transcribed to cDNA for stability and then fragmented and tagged with specific sequences that allow for batch processing of samples. 3) NGS: Samples are loaded onto a specialized flow cell where amplification and sequencing will take place. Cluster generation forms millions of “DNA colonies” and sequencing-by-synthesis begins to record the sequence of base pairs in each fragment. 4) Bioinformatic analysis: Once sequences are generated, they are aligned to a reference genome, normalized and process for quality control. A host of different analyses can then be applied depending on the outcome of interest. Readers are encouraged to access publicly available NGS datasets and bioinformatics resources for further information. RPKM: reads per kilobase of transcript per million mapped reads; FPKM: fragments per kilobase of transcript per million mapped reads; CPM: counts per million reads mapped; TPM: transcripts per million reads mapped.

Figure 2.. CRISPR/Cas9 Gene Editing and Next Generation CRISPR Technologies.

A. Short guide RNA (sgRNA) associated with Cas9 pairs with a complementary genomic sequence that is 5’ to a protospacer adjacent motif (PAM.) Because of the genomic target sequence’s proximity to the PAM, the Cas9 cleaves the DNA, creating a double-stranded break (DSB). Repair of the DSB occurs through one of two endogenous processes, non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ can result in short insertions or deletions (indels) of sequence at the break site. Homology-directed repair uses template DNA; if exogenous template DNA is provided that is homologous to the target sequence with a desired sequence inserted at the break site, this directed insertion can be incorporated into the genomic DNA. B. Potential next generation CRISPR technology that has yet to be implemented includes inducible (top) and conditional (bottom) single-base editing without DSB. The example given is a method of inducible base-pair editing using a tetracycline-dependent promoter governing Cas9-nickase fused with a deaminase and guide RNA. The bottom example of conditional single-base editing is the same nCas9-deaminase and sgRNA in reverse orientation with flanking LoxP sites, requiring the presence of Cre recombinase to flip the orientation of the sequence and allow for transcription and translation.

Figure 3:. Inducible pluripotent stem cells overview:

Human cardiac and brain tissue is difficult to access and usually not available for drug testing. After reprogramming mature, somatic cells via induction of four transcription factors (Yamanaka factors, Oct4, Sox2/4, Klf4, and Myc), into inducible pluripotent stem cells (iPSC) can be differentiated by modifying cell culture conditions and adding cell-type specific differentiation factors or small molecules into cardiovascular tissue (EC: endothelial cell; CM: cardiomyocytes; CFB: cardiac fibroblasts) or neuronal tissue (Neuro: neurons; AstrC: astrocytes; OligoD: oligodendroglia cells; PeriC: pia cells; MG: microglia cells). Complexity of development and testing increases with the development of organoids or complex engineered tissues. Each tissue type can be subject to functional, morphological, or genetic studies. (scRNA-seq= single cell RNA sequencing).