Basic Science

E   Narrative Review Article

A Guide to Understanding “State-of-the-Art” Basic

Research Techniques in Anesthesiology
Detlef Obal, MD, PhD, DESA,*† Shaogen Wu, MD, PhD,* Andrew McKinstry-Wu, MD,‡ and
Vivianne L. Tawfik, MD, PhD*

Perioperative medicine is changing from a “protocol-based” approach to a progressively person-

alized 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
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significantly evolved in recent years with the advent of powerful approaches that have transla-
tional relevance. It is incumbent on us as a primarily clinical specialty to have an in-depth under-
standing 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 tech-
niques: 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 physi-
cians when confronted with basic science articles and encourage investigators to apply “state-
of-the-art” technology to their future experiments.  (Anesth Analg 2020;131:450–63)

GLOSSARY
2D = 2 dimensional; AMPAR = α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor; AstrC
= astrocytes; Cas = CRISPR-associated proteins; cDNA = complementary DNA; CFB = cardiac
fibroblasts; CM = cardiomyocytes; c-Myc =  cMYC oncogene; CPM = counts per million reads
mapped; CRISPR = clustered regularly interspaced short palindromic repeat; crRNA = CRISPR
RNA sequence; DEG = differentially expressed genes; dNTP = deoxynucleoside triphosphate; DSB
= double-stranded DNA break; dsDNA = double-stranded DNA; EC = endothelial cell; EGFR =
epithelial growth factor receptor; EHT = engineered heart tissue; ESC = embryonic stem cell; FACS
= fluorescence-activated cell sorting; FDR = false discovery rate; FFPE = formalin-fixed paraffin-
embedded; FPKM = fragments per kilobase of transcript per million mapped reads; GO = Gene
Ontology; GSEA = gene set enrichment analyses; HDR = homology-directed DNA repair; hESC =
human embryonic stem cell; hiPSC = human iPSC-derived cell; hiPSC-CM = human iPSC-derived
cardiomyocyte; hiPSC-NC = human iPSC-derived neuronal cells; iPSC = inducible pluripotent stem
cell; KEGG = Kyoto Encyclopedia of Genes and Genomes; Klf4 = Krüppel-like factor 4; LCM = laser-
capture microdissection; LoxP = 34 bp sequence where Cre recombinase binds; MG = microglia
cells; nCas9 = Cas9 nickase; Neuro = neurons; NGG = 3 bp sequence with any nucleotide (N)
followed by 2 guanines (GG); NGS = next-generation sequencing; NHEJ = nonhomologous end
joining; Oct2/4 =  octamer-binding transcription factor 2 and 4; OligoD = oligodendroglia cells;
PAMs = protospacer adjacent motifs; PeriC = pia cells; PGC-1α = peroxisome proliferator-activated
receptor-𝛾-coactivator 1-α; QC = quality control; RNA-seq = RNA sequencing; RPKM = reads per
kilobase of transcript per million mapped reads; rtTA = reverse tetracycline-controlled transactivator;
scRNA-seq = single-cell RNA sequencing; sgRNA = single-guide RNA; SIRS = systemic inflammatory
response syndrome; Sox2 = SRY (sex determining region Y)-box 2 transcription factor; TALEN =
transcription activator-like effector nucleases; tet = tetracycline; TPM = transcripts per million; TRE
= tetracycline-responsive element

From the *Department of Anesthesiology, Perioperative, and Pain Medicine,  Grant K08NS094547 (V.L.T.), K08GM123317 (A.M.-W.), and the Rita Allen
Stanford University School of Medicine, Stanford, California; †Department
of Anesthesiology, Perioperative, and Pain Medicine and Cardiovascular
Institute, Stanford University School of Medicine, Stanford, California; and
‡Perelman School of Medicine, University of Pennsylvania, Philadelphia,
Pennsylvania.
Accepted for publication March 4, 2020.
Funding: D.O. was supported by a Seed Grant of the Stanford CV Institute
and the Transdisciplinary Initiatives Program (TIP) of the Stanford Maternal
& Child Health Research Institute, institutional and/or departmental
Copyright © 2020 International Anesthesia Research Society
DOI: 10.1213/ANE.0000000000004801
resources, as well as grant funds from the National Institutes of Health (NIH)
Foundation Award in Pain (V.L.T.).
The authors declare no conflicts of interest.
Portions of the content of this review have been presented in seminar format on
Monday, May 20, 2019 at the IARS annual meeting in Montreal, QC, Canada.
Listen to this Article of the Month podcast and more from OpenAnesthesia.org®
by visiting http://journals.lww.com/anesthesia-analgesia/pages/default.aspx.
Reprints will not be available from the authors.
Address correspondence to Detlef Obal, MD, PhD, DESA, Department of
Anesthesiology, Perioperative and Pain Medicine, Stanford University, 300
Pasteur Dr, Palo Alto, CA 94305. Address e-mail to obal@stanford.edu.

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 EE Narrative Review Article
NEXT-GENERATION SEQUENCING FOR
UNDERSTANDING CELL-SPECIFIC CONTRIBUTIONS
TO YOUR DISEASE OF INTEREST
In 1977, Sanger et al1 developed a sequencing tech-
nique based on the incorporation of terminal dide-
oxynucleotides that expedited the sequencing of short
DNA segments. “Sanger sequencing” has been widely
used to identify gene mutations as direct causes or
susceptibility factors associated with the development
of genetic disorders; however, sequencing occurs 1
DNA fragment at a time, limiting scalability. During
the past decade, next-generation sequencing (NGS)
approaches using sample multiplexing (multiple
samples in 1 sequencing run) have been developed
that allow for high-throughput screens for genomic
mutations and quantification of gene expression in
tissue or cells from a disease of interest.2 Unlike previ-
ous assays, NGS techniques directly read sequences
of a pool of gene fragments allowing for the detec-
tion of novel transcripts, providing unbiased and
comprehensive genomic coverage.3 As a result, NGS
approaches have truly revolutionized discovery in a
huge variety of science fields (see Figure 1).
DESCRIPTION OF THE TECHNIQUE
RNA sequencing (RNA-seq) is the dominant NGS
approach used in basic and clinical research. In this
review, we will focus on RNA-seq to illustrate the
workflow of an NGS-based approach and discuss its
advantages and limitations. The overall workflow
consists of 4 parts: (1) preparation of samples, (2)
library construction, (3) NGS, and (4) bioinformatic
analysis.
Preparation of Samples
The starting materials for RNA-seq study are diverse
including fresh clinical samples, laser-capture micro-
dissection (LCM)–excised sections from formalin-fixed
paraffin-embedded (FFPE) tissue, or cells obtained
using fluorescence-activated cell sorting (FACS).
A standard protocol for tissue collection and RNA
purification is critical for successful transcriptomic
profiling because the degradation of RNA can intro-
duce biological bias.4 Collecting fresh tissue or cells
in a commercially available RNA stabilization solu-
tion is a practical way to prevent RNA degradation.
Newer “single-cell” approaches dissociate tissue into
single-cell suspensions that allow the transcriptome of
individual cells to be determined. However, these pro-
tocols can also cause cellular stress and transcriptomic
changes,5,6 a fact that must be taken into account when
designing and interpreting such studies.
Library Construction
This step converts the sample into a sequencing library
that can be sequenced (read) on an NGS instrument.
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The majority of RNA-seq first requires the prepara-
tion of a complementary DNA (cDNA) library for
stability. The selection of a specific protocol for sub-
sequent library construction depends on the purpose
of the study (eg, a ribosomal RNA depletion protocol
allows detection of long noncoding RNAs that lack
poly-A tails).7 In general, library preparation involves
random fragmentation of the cDNA sample into
shorter segments that can be reliably sequenced then
followed by ligation of specialized 3′ and 5′ adapters
that barcode samples and bind to the NGS flow cell
(see below).8
Next-Generation Sequencing
Out of the many types of NGS techniques in the mar-
ket,9 the most popular one is termed “sequencing by
synthesis.”10 Generally speaking, each library, con-
sisting of cDNA fragments labeled with adapters, is
loaded onto a specialized flow cell with several lanes
where amplification and sequencing will take place.
Along each lane, there are millions of complementary
oligonucleotides that anchor the libraries to the flow
cell. Once the fragments have attached, a phase called
cluster generation begins to form millions of “DNA
colonies.” Next, primers and DNA polymerase start
adding 1 fluorescently tagged deoxynucleoside tri-
phosphate (dNTP) base,11 and in each round of syn-
thesis, the sequencer records the base added to each
fragment on the flow cell in parallel.
There are 3 major configurations of NGS which
should be carefully chosen depending on the study
purpose: read length, strand specificity, and sequencing
depth. Read length refers to how many nucleotides can
be read from a given library fragment. Longer reads can
increase the specificity but depend on sequencer type
and may cost more.9 Paired-end sequencing allows for
both ends of the DNA fragment to be sequenced and
can increase precision at splicing junctions,12 repeti-
tive areas of the genome, or other difficult-to-sequence
segments. Strand specificity refers to sequencing that
retains the orientation of the transcripts from 5′ to 3′
end. This technique further increases the accuracy of
the results by ensuring that reads are mapped to the
correct gene and not a similar gene going in the oppo-
site direction.13 Sequencing depth refers to how many
reads (fragments) are sequenced per sample. A total
read depth of 10 to 30 million reads per sample is con-
sidered “deep” enough for the coverage of larger tran-
scriptomes such as human or mouse to ensure that low
abundant transcripts are detected.9
Bioinformatic Analysis
The output of the NGS process will be millions of
sequences that are generated, processed, and assigned
to each sample, resulting in sequence data on the order
of gigabytes. The first step of RNA-seq data analysis
 Basic Research Techniques in Anesthesiology

Figure 1. NGS overview. (1) Preparation of samples: Multiple different types of samples can be processed to obtain material for NGS. RNA is
extracted from bulk, LCM or 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. cDNA indicates complementary DNA;
CPM, counts per million reads mapped; FACS, fluorescence-activated cell sorting; FPKM: fragments per kilobase of transcript per million mapped
reads; LCM, laser-capture microdissected; NGS, next-generation sequencing; RPKM, reads per kilobase of transcript per million mapped reads;
TPM, transcripts per million reads mapped.

should be the assessment of the raw sequences. FastQC
is a quality control (QC) tool which provides an over-
view of raw RNA-Seq data (Babraham Bioinformatics,
https://www.bioinformatics.babraham.ac.uk). To fur -
ther improve the RNA-seq data quality, tools (eg,
Trimmomatic, http://www.usadellab.org/cms/?
page=trimmomatic) can be used for trimming and
removal of library adapters that were placed for bind-
ing to the flow cell.14 After QC, sequenced reads are
mapped, that is, aligned, to a reference genome or to
a transcriptome database. The mapped reads are then
counted and normalized for differential expression.

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 EE Narrative Review Article
Fragments per kilobase of transcript per million
mapped reads (“FPKM”)/reads per kilobase of tran-
script per million mapped reads (“RPKM”) or counts
per million reads mapped (“CPM”) are some of the
units used to calculate the abundance of each gene
expressed in a sample using different algorithms.15
Transcripts per million (“TPM”) is now becoming
popular, which makes it easier to compare the propor-
tion of reads that are mapped to a gene in each sample
as it first normalizes for gene size followed by read
depth.16,17
The most powerful use of RNA-seq is in finding dif-
ferentially expressed genes (DEGs) between ≥2 condi-
tions. There are many tools that perform differential
expression calculations including DESeq, edgeR, and
Limma-Voom, all of which are available through R/
Bioconductor.18 Most tools use regression or nonpara-
metric statistics to calculate a false discovery rate (FDR)
for multiple hypotheses and identify DEGs by log fold
change and statistical significance. To obtain a higher-
level biological understanding of a list of DEGs, genes
can be annotated using Gene Ontology (GO)19 or Kyoto
Encyclopedia of Genes and Genomes (KEGG) terms,20
which categorize genes based on the family they belong
to (eg, “inflammatory response” or “immune system
process”). Downstream gene set enrichment analyses
(GSEA) can then determine whether there are certain
pathways or terms (eg, “circadian rhythms” or “plate-
let activation”) that are overrepresented in experimen-
tal versus control samples.21 Notably, there are many
useful web-based NGS analysis tools available for
analysis and visualization of large genomic data (see
Figure 3). However, we recommend consultation with
an experienced bioinformatician to ensure that appro-
priate QC is performed, to reduce the risk of bias, and
to increase the reproducibility of the results.
ADVANTAGES AND LIMITATIONS OF NGS
TECHNOLOGY
NGS techniques, such as RNA-seq, have some clear
advantages over older approaches including higher
throughput, increased accuracy, ability to detect novel
transcripts, higher sensitivity and specificity, and abil-
ity to detect low-abundance transcripts.22,23 NGS also
seems to be the most cost-efficient tool for genome/
transcriptome study nowadays. For example, sequenc-
ing the entire human genome by NGS now costs no
more than $1000, compared to $100 million in 2001.
However, NGS has clear limitations that must be taken
into account when designing and interpreting experi-
ments. First, transcription and translation occur at vari-
able rates as does degradation of mRNA and protein.
As a result, RNA levels may not necessarily reflect
the actual level or activity of proteins of interest. This
highlights the need for validation of DEGs at the pro-
tein level after RNA-seq profiling. Second, methods of
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sample preparation may trigger certain cellular path-
ways (eg, stress responses) which may be falsely over-
represented in the final dataset.24 Third, batch effects
may skew results and experiments need to be care-
fully designed to mitigate them. Finally, NGS requires
a large platform and supercomputer, which might not
be easily accessible to all researchers. Importantly, sev-
eral NGS sequencing services have recently entered the
market at a reasonable cost and can be a good choice if
internal core facility access is limited.
IMPLICATIONS FOR ANESTHESIOLOGISTS
In clinical anesthesiology, NGS can be used for the
identification of biomarkers for intervention, treatment
outcome prediction, and understanding disease sus-
ceptibility, among other applications.25,26 For example,
Tsalik et al25 sequenced peripheral blood RNA of 129
representative subjects with systemic inflammatory
response syndrome (SIRS) or sepsis. They found that
the expression of 338 genes differed between subjects
with SIRS and those with sepsis and the expression
of 1238 genes differed with sepsis outcome (survival
versus nonsurvival).25 They also discovered that the
expression of a gene called VPS9D1, which may con-
trol cell signaling through endocytosis of intracellu-
lar receptors, increased in sepsis survivors, who also
expressed a higher number of missense variants in
this gene.25 Another RNA-seq study relevant to our
specialty examined ischemic changes, induced by cold
blood cardioplegia on the left ventricular myocardium
in 45 patients undergoing aortic valve replacement.26
Through transcriptomic analysis, 1241 DEGs were
identified when comparing baseline samples to those
obtained 79 minutes after aortic cross-clamping.26
Further functional study of these candidate genes may
provide greater insight into the pathophysiology of
ventricular ischemia that will guide the development
of cardioprotective strategies.26
FUTURE DIRECTIONS OF NGS
Many different NGS variants (eg, single-cell RNA-
seq,27,28 spatial transcriptomics,29,30 and 16S rRNA
sequencing for microbiome study)31 have been devel-
oped. These novel NGS techniques allow researchers to
look at the transcriptome heterogeneity and the spatial
arrangement of cell types in a given tissue. Using NGS,
we are able to uncover new genes of interest but also to
cluster DEGs within key pathways that may be targeted
for improved therapeutic efficacy. Furthermore, as
additional biomarkers are identified, there is power to
monitor disease progression and treatment outcomes.
NGS also provides a cost-efficient tool for personalized
medicine approaches by identifying whole transcrip-
tome signatures that may allow for mechanism-based
treatments. With the development of standardized
laboratory protocols and bioinformatics pipelines, NGS
 Basic Research Techniques in Anesthesiology
is now becoming more accessible for use in both basic
and clinical research. Finally, several publicly avail-
able, user-friendly NGS data repositories exist that can
be interrogated for hypothesis development or used to
cross-reference new datasets.32 Overall, NGS technolo-
gies have already contributed new knowledge to our
field, and with further adoption by anesthesiologist sci-
entists, there lies the huge potential for furthering our
impact on a diverse set of disease-specific questions.
CLUSTERED REGULARLY INTERSPACED SHORT
PALINDROMIC REPEAT: A VERSATILE TOOLBOX
FROM GENE EDITING TO EXPRESSION
Precise genome editing has become an indispens-
able tool for research programs incorporating animal
models and is increasingly finding use in translational
research. The clustered regularly interspaced short pal-
indromic repeat (CRISPR) toolset is a highly adaptable,
easily implemented approach for changing a genome
at a precise, specified location (ie, directed recombina-
tion). Anesthesia research has incorporated gene edit-
ing using various techniques for decades,33–36 and as
CRISPR has become the predominant method of edit-
ing, it has become central to many anesthesia research
endeavors. Uses for this versatile toolset have already
extended to include gene regulation, single-base edit-
ing, and inducible recombination. The future will
undoubtedly see a continued expansion of CRIPSR
technology with innovations such as conditional or
inducible single-nucleotide editing on the horizon.
While genetic recombination in mammalian mod-
els was pioneered nearly 35 years ago,37 the feasibil-
ity and adoption of this approach have dramatically
increased over the past decade. Early genetic editing
relied on rare spontaneous recombination events to
incorporate introduced genetic material after untar-
geted double-stranded DNA (dsDNA) breaks at
random locations. Eukaryotes use 2 separate pro-
cesses to repair such breaks: homology-directed DNA
repair (HDR) or nonhomologous end joining (NHEJ).
NHEJ is the faster and more common repair mecha-
nism and involves directly ligating 2 broken ends of
DNA together without the use of any DNA template.
This type of repair, as it does not have a template to
error check, can result in insertions or deletions at the
break site. HDR, in contrast, does use a DNA tem-
plate to facilitate and error check the repair process
(Figure  2A).38 By introducing template DNA with a
desired change rather than the native genome, which
would normally serve as a template, HDR can be used
to specifically alter genomic DNA.
Relying on spontaneous recombination on non-
targeted breaks is extremely inefficient with a high
chance of gene incorporation at off-target sites.39,40
The ability to specifically target dsDNA cleavage
increases both the reliability and specificity of genome
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editing. This is despite most double-stranded breaks
being repaired using NHEJ rather than the desired
template-driven HDR.38 The ability to adaptably tar-
get DNA breaks expanded the adoption of genome
editing due to an increased rate of recombination
at desired loci. Targeted breaks were first produced
using mega-nucleases, but poor flexibility in target
sequence selection of these large proteins caused
them to be replaced by chimeric proteins fusing
DNA recognition domains with the DNA cleavage
domain of the endonuclease Fok I.41,42 This technique
requires engineering new chimeric proteins for each
new sequence to be targeted. The technical hurdles
involved in engineering and cloning novel proteins
limited the adoption of techniques using this strategy.
ADVANTAGES AND LIMITATIONS OF CRISPR
CRISPRs major advantages over its gene-editing pre-
decessors are its simplicity and flexibility, while it
largely matches the efficacy and efficiency of earlier
techniques. The crucial elements of the CRISPR tool-
box are derived from an endogenous bacterial antivi-
ral process. In this bacterial system, 2 short CRISPR
RNA sequences (crRNAs)—one of which includes
a 17–28 nucleotide portion homologous to bacterio-
phage sequences—form complexes with CRISPR-
associated proteins (Cas) to target the viral sequence
encoded by the crRNA.43 When such a complex incor-
porates a DNA-cleaving Cas element, such as Cas9,
the result is a targeted double-stranded DNA break
(DSB) with the target specified by an RNA sequence.
For the CRISPR-Cas complex to attach and cleave the
viral DNA, the targeted viral genomic sequence must
include a short protospacer adjacent motif (PAM), a
3–7 nucleotide long sequence specific to a given Cas
enzyme, 3' to the targeted sequence. Though these
systems are native to bacteria and are intended to tar-
get viral DNA sequences, with modifications to Cas9
to include a nuclear localization sequence, they are
able to modify mammalian genomic DNA as well.44–
46
Another early modification to the system was the
substitution of a single-guide RNA (sgRNA) for the 2
crRNAs, further reducing complexity.47 The modular
nature of the system, with interchangeable Cas pro-
tein elements (for enzymatic activity) and sgRNAs
(for target sequence selection) combined with the
comparative ease of nucleotide synthesis, makes this
system an extremely useful biotechnology tool, both
flexible in its application, and simple enough to imple-
ment that it can be readily adopted without extensive
technical prerequisites.
CRISPR/Cas9 was the first widely adopted
CRISPR toolset for gene editing. Despite its simplicity
and flexibility, this approach is not without its draw-
backs. While RNA-mediated targeting for CRISPR/
Cas9 is flexible and easily changed, genomic locations
ANESTHESIA & ANALGESIA
 EE Narrative Review Article

Figure 2. CRISPR/Cas9 gene-editing and next-generation CRISPR technologies. A, sgRNA associated with Cas9 pairs with a complementary
genomic sequence that is 5′ to a PAM. Because of the genomic target sequence’s proximity to the PAM, the Cas9 cleaves the DNA, creating a
DSB. Repair of the DSB occurs through 1 of 2 endogenous processes, NHEJ or HDR. NHEJ can result in short insertions or deletions (indels)
of sequence at the break site. HDR 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 deami-
nase 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 trans-
lation. Cas9 indicates CRISPR-associated protein 9; CRISPR, clustered regularly interspaced short palindromic repeat; DSB, double-stranded
DNA break; HDR, homology-directed repair; LoxP, 34 bp sequence where Cre recombinase binds; nCas9, Cas9 nickase; NHEJ, nonhomologous
end joining; PAM, protospacer adjacent motif; rtTA, reverse tetracycline-controlled transactivator; sgRNA, single-guide RNA; tet, tetracycline;
TRE, tetracycline-responsive element.

for cleavage are limited by the necessity of a PAM
sequence 3′ to the cleavage site. PAM sequences are
short and occur frequently enough within the genome
that this restriction rarely presents a major impedi-
ment, but it is a limitation. Like their predecessors,
zinc-finger nucleases and transcription activator-like
effector nucleases (TALENs), CRISPR/Cas9 sys-
tems make use of endogenous DNA repair processes
after DSBs. The 2 separate events—the initial DSB
and subsequent repair—are each possible sources of

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 Basic Research Techniques in Anesthesiology
error. Though targeting is specified with PAMs and
the 17–20 nucleotide sequence in crRNA, careful
examinations of off-target effects show that CRISPR
systems display some flexibility in their targeting
of the sequence, causing DSBs at sites not predicted
by sequence alone.48,49 These off-target cleavages can
produce insertions and deletions at disparate sites
within the genome. Even when a DSB occurs in the
correct location, NHEJ, with its associated insertions
of random genetic material or deletions of proxi-
mate sequence, occurs 8 times more frequently than
the more desirable HDR.50 Numerous improvements
have made to CRISPR/Cas genome-editing systems,
largely focused on increased targeting flexibility,
decreased off-target cutting, and increased rate of
HDR over NHEJ after a cut. Increased targeting flex-
ibility through altering PAM sequences is a signifi-
cant source of innovation. The most commonly used
Cas9—derived from Streptococcus pyogenes—has 3
bp sequence with any nucleotide (N) followed by 2
guanines (GG) (NGG) as its PAM sequence, but Cas
derived from other species have alternate sequences,
expanding potential targets within a genome. In addi-
tion to naturally occurring homologs, engineered Cas
variants can have altered, and even more flexible,
PAM requirements.51–53 Diverse strategies have been
successful at decreasing the rate of off-target DSBs,
another goal for innovation. Cas variants specifically
engineered to decrease the rate of off-target cutting,54
altering sgRNA length, or delivering the complete
sgRNA–Cas ribonucleoprotein complex (rather than
a plasmid encoding both) have all succeeded at reduc-
ing off-target cutting.55,56 Using Cas nickases, which
cut only 1 of the 2 DNA strands, and 2 sgRNAs inde-
pendently targeting sequences directly opposite
each other, 2 cuts need to independently occur to
create a DSB, significantly reducing the rate of off-
target DSBs.57 Finally, temporal and spatial control
of enzymatic activity using light- or small-molecule-
activated Cas can also decrease off-target cutting.58,59
Similarly, diverse approaches have been deployed
toward increasing the rate of template-driven HDR
and decreasing the incidence of NHEJ after producing
a DSB. The rate of HDR can be increased using either
small molecule adjuvants or single-stranded DNA
templates.60,61 Certain Cas variants produce staggered
DSBs in DNA, leaving overhanging sticky ends. These
staggered breaks have a higher rate of HDR, such that
use of this class of Cas, including Cpf1, can increase
the rate of successful recombination.62
Applications of CRISPR-based systems have
expanded beyond simple facilitated recombination
using targeted DSBs. Fusing catalytically inactive Cas9
(dCas9) to other functional domains has emerged as a
flexible means of targeting specific loci in the genome
for a variety of purposes. These include attaching gene
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regulatory elements to dCas9 to enhance or inhibit tran-
scription,63,64 fluorescent tags to visualize gene locations
in the nuclei of live cells,65 and methyltransferase to
alter local DNA methylation.66 Newer techniques have
opened the door to gene editing without creating DSBs.
The fusion of Cas nickase with various deaminases
allows the direct conversion of C·G > T·A or A·T > G·C
and removal of the opposite paired base such that, by
targeting the correct strand, any single genomic base
pair can be converted to any other.67,68 This technology
is still in its early stages, however, and deaminases can
induce significant off-target mutations.69 The potential
for such “second-generation” CRISPR-based gene edit-
ing nevertheless has yet to be fully realized. Specifically,
approaches that have been previously used in conjunc-
tion with first-generation CRISPR gene-editing tech-
nology, inducible and conditional expression, could be
applied to second-generation systems to produce cell-
line-specific or inducible targeted single-base muta-
tions (Figure 2B) Such limited targeted mutations could
allow for a range of studies on critical genes that oth-
erwise prove lethal when altered during development.
IMPLICATIONS FOR ANESTHESIOLOGISTS
The use of CRISPR technology for gene editing has
only become widely adopted over the past 5 years,
and thus we are just beginning to see studies using
CRISPR-engineered mutants in the anesthesiol-
ogy literature.70 As CRISPR-based gene-editing and
advanced CRISPR techniques become the foundation
for a wide variety of investigations in the science and
practice of anesthesiology, it will become ever more
critical for anesthesiologists to understand many of
the previously mentioned hazards and potential limi-
tations of the technique, in addition to appreciating
the diverse applications. In interpreting these types of
studies, it is important that readers look for appropri-
ate controls accounting for potential off-target cutting
including insertions and deletions. Alternatively, the
study authors may include measures to minimize the
impact of such events such as backcrossing animals
to a wild-type background or the use of enzymes or
adjuvants that minimize such off-target events.
FUTURE DIRECTIONS OF CRISPR
CRISPR-based technologies have significantly low-
ered the barrier to entry for gene-editing approaches,
and new applications offer similar potential for gene
regulation and epigenomic approaches. CRISPR has
already broadened the types of basic science ques-
tions that can be asked by opening the door to the use
of new model organisms.71 Separately, second-gener-
ation editing has the potential to bridge gene-editing
technologies into the clinical realm in the coming
years through precise and limited genomic altera-
tions. We have yet to see the full scope of the impact
ANESTHESIA & ANALGESIA
 EE Narrative Review Article
of these powerful and flexible approaches, but con-
tinued refinement with reduction of off-target effects
will be necessary before this technology can move
from the bench to the bedside.
INDUCIBLE PLURIPOTENT STEM CELL
TECHNOLOGY: A ROAD TO PERSONALIZED
MEDICINE
Human embryonic stem cells (hESCs) are derived
from the inner cell mass of fresh or frozen embryos
at the blastocyst stage of development.72 Embryonic
stem cells (ESCs) are self-renewable and able to give
rise to cells of all 3 germ layers (ectoderm, endoderm,
and mesoderm).73,74 Indefinite replication makes
hESCs a valuable tool to study anesthetic mechanisms
in human tissue. However, ethical controversies and
limited supply of donor human embryos restricted
the use of this cell type and alternative approaches
were warranted.
In 2006, Takahashi and Yamanaka75 performed an
experiment in which they selected 24 different tran-
scription factors as candidates to induce pluripotency
in somatic cells, that is, reprogramming of already
differentiated cells into ESC-like cells. After exten-
sive screening, they identified 4 transcription  factors
essential to produce ESC-like colonies able to form
teratoma: octamer-binding transcription factor 2 and 4
(Oct2/4), SRY (sex determining region Y)-box 2 (Sox2),
Krüppel-like factor 4, (Klf4), and the oncogene cMYC
(c-Myc) (also known as “OSKM”).75 These inducible
pluripotent stem cells (iPSCs) have become a valuable
tool in basic science, and only 6 years after his origi-
nal observation, Shinya Yamanaka (together with John
B. Gurdon) received the Nobel Prize in Physiology
or Medicine for their “discovery of reprogramming
mature cells into pluripotent stem cells.”
The great potential of this technique relies on its
ability to generate any somatic cell line out of an indefi-
nitely dividing stem cell pool, specific to a particular
patient and collectable without invasive procedures.
In other words, peripheral human blood cells can be
reprogrammed into stem cells which will be subse-
quently differentiated into a somatic cell of any type.76–81
With regard to morphology, surface marker expression,
global gene expression profile, DNA methylation sta-
tus, and other characteristics, human iPSC-derived cell
(hiPSC) and hESC are considered to be similar.82 Herein
we will focus on hiPSC-derived somatic cells, which are
of particular interest for anesthesiologists: cardiomyo-
cytes and neurons (Figure 3). Although hiPSC-derived
somatic cells are frequently cultured in monolayers
(2-dimensional [2D] culture) when used for drug and
phenotype testing, our description will concentrate on
hiPSC-derived cardiomyocyte (hiPSC-CM) and neuro-
nal cell (hiPSC-NC) tissues grown as organoids (ie, 3D
August 2020 • Volume 131 • Number 2 www.anesthesia-analgesia.org 457
Copyright © 2020 International Anesthesia Research Society. Unauthorized reproduction of this article is prohibited.
structures), which are build out of different cell types to
better reflect the “natural” original.
To build organoids based on patients’ own cells has
been a long-term objective within the iPSC commu-
nity; however, the organoids should reflect “structure”
and “cell composition” of their natural counter-
parts. Nowadays, structure modifications have been
archived by applying different bioengineering meth-
ods83,84 and by modifying cell culture conditions.85,86
Diversity of cell composition within the organoids
has been realized in 2 different ways: human iPSC/
ESC have been differentiated by stepwise adding
small molecules and growth factors resulting in sub-
sequent development of various cell types reflecting
“naturally” formed organoids.87 Alternatively,  hiP-
SCs have been differentiating into multiple cell types
separately and then in a subsequent step “assembled”
to the final organoid. While the first method allows
to study “natural” progression of cell differentiation
during organogenesis, the second method allows to
control for cell-type composition within the organoids
superior for description of cell–cell interactions.
Compared to 2D cell layers, hiPSC growing as
3D organoids have less contact with culture plates,
thus favoring interaction between cells and main-
taining histological and genetic complexity even
after long-term culture.88 Importantly, cells grown
in organoids have a distinct gene expression profile
compared to their 2D counterparts resulting in con-
trasting pharmacological profiles. Luca et al89 dem-
onstrated that tumor cells cultured in a 3D model
express significantly less epithelial growth factor
receptors (EGFRs, a factor important for controlling
cell proliferation) compared to tumor cells cultured
in 2D culture which made these cells less suscep-
tible to EGFR tyrosine kinase inhibitors. As EGFR
tyrosine kinase inhibitors are tested as chemothera-
peutic agents, the therapeutic efficiency reported
in different studies may deviate significantly and
impact drug development.
HiPSC-DERIVED CARDIAC ORGANOIDS
By aligning hiPSC-CM in parallel rather than ran-
domly within an extracellular matrix,90 Zimmermann
et al,91 and Eschenhagen et al92 developed engineered
heart tissue (EHT) models in which cardiomyocytes
increased in size and started to form a connected syn-
cytium. EHT allows advanced functional assessment
of human tissue in a dish,92 even in a high-throughput
screening format.93 The current end of these bioen-
gineering developments constitutes a 3D electrome-
chanically coupled, fluid-ejecting miniature ventricle
derived from hiPSC-CM,94 in which anesthetic agents
and other drugs might be tested under “physiologi-
cal” conditions “in vitro” in the future.
 Basic Research Techniques in Anesthesiology

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 4 transcription factors (Yamanaka factors, Oct4, Sox2/4, Klf4, and Myc)
into iPSC can be differentiated by modifying cell culture conditions and adding cell-type specific differentiation factors or small molecules into
cardiovascular tissue (EC, CM, CFB) or neuronal tissue (Neuro, AstrC, OligoD, PeriC, MG). 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. AstrC indicates astrocytes; CFB, cardiac fibroblasts; CM, cardiomyocytes; EC, endothelial cell; iPSC, inducible pluripotent stem cells;
Klf4, Krüppel-like factor 4, transcription factor; MG, microglia cells; Myc, MYC proto-oncogene, transcription factor; Neuro, neurons; Oct4,
octamer-binding transcription factor 4; OligoD, oligodendroglia cells; PeriC, pia cells; scRNA-seq, single-cell RNA sequencing; Sox2/4, SRY (sex
determining region Y)-box 2 transcription factor.

HiPSC-DERIVED CEREBRAL ORGANOIDS their modes of action. Currently, the time to gener-
HiPSC-derived cerebral organoids offer great poten- ate self-organized 3D structures spans from 50 to 100
tial to study the neurotoxic effects of anesthetics and days.95,96 The construction of neuronal organoids97,98

458   
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Copyright © 2020 International Anesthesia Research Society. Unauthorized reproduction of this article is prohibited.
 EE Narrative Review Article
allows the prediction of anesthetic-induced neuro-
toxicity,99 and with advancements in single-cell tech-
nology, we have the tools to obtain transcriptomes of
single cells and to cluster cells with identical transcrip-
tional activities.100 With the combination of hiPSC-
NC and vascular cells, neuronal-vascular units have
been developed101,102 to study brain development103
or function and neurotoxicity of anesthesia-inducing
drugs.104,105 Unfortunately, this technique is still unde-
rutilized in our specialty and during anesthetic and
analgesic drug development.
ADVANTAGES AND LIMITATIONS OF iPSC
TECHNOLOGY
Although hiPSC have been discovered >10 years ago,
the technique is still evolving and some limitations
should be considered. Retrieving cells from patients
appears to be relative easy and straightforward, how-
ever, as the efficiency of reprogramming primary cells
(skin fibroblast versus blood cells) depends on the cell
type,106 which should therefore be carefully chosen.
Another consideration for using hiPSC technology
in clinical practice may be the maturation status of
hiPSC-derived cardiomyocytes or neurons. Although
functional cardiomyocytes (ie, contracting cells,
hiPSC-CM) develop within 7–9 days,107 an additional
of 90–120 days may be required to generate cardiomy-
ocytes with a more mature phenotype (ie, expression
of adult genes, normal morphology, and expres-
sion of normal T-tubule and sarcomere structure).108
Similarly, differentiation hiPSC toward neuronal tis-
sue requires between 2109–111 and 6 weeks.112–114 These
culture requirements may currently not be suitable
for “preoperative” bedside tests but may impact care
for patients undergoing long-term planned elective,
high-risk procedures or for patients with repeated
anesthetic exposures (ie, pediatric patients after cor-
rection of congenital heart defects).
Additional concerns about hiPSCs genetic stabil-
ity during reprogramming as well as differentiation-
dependent variability and heterogeneity exist.115,116
These reservations may be justified; however, they
reflect limitations of a relatively young technology
which will be resolved within the coming years.
IMPLICATIONS FOR ANESTHESIOLOGISTS
How do hiPSC-derived cells help anesthesiologists to
understand cellular function and where are the limi-
tations? By using a few assays as an example, advan-
tages and limitations of iPSC-based techniques will be
outlined.
One of the advantages of hiPSC-CM and hiPSC-
NC is the ability to perform functional assessments
of patient-specific tissue in vitro. Cardiac arrhyth-
mias and QTc interval prolongation are significant
milestones to overcome during drug development.
August 2020 • Volume 131 • Number 2 www.anesthesia-analgesia.org 459
Copyright © 2020 International Anesthesia Research Society. Unauthorized reproduction of this article is prohibited.
With the development of hiPSC-CM, drug-induced
QTc prolongation can be successfully studied in
human tissue “in a dish.” hiPSC-CM of patients
with inherited channelopathies can be easily gen-
erated from a 10-mL blood sample to study ion
channel function or membrane potential changes
by different methods (ie, patch clamp, fluorescence
dyes, genetically introducing voltage, and/or cal-
cium indicators117–120).
Most recently, hiPSC-CM has been used to determine
the cardiac side profile of KSEB01-02, a new compound
with hypnotic properties.121 Using a high-throughput
screening method, KSEB01-02 turned out to be less car-
dio-depressant than propofol, elucidating the advan-
tage of using hiPSC-CM to screen for cardiovascular
side effects of a new drug in human heart tissue.
Similar methods can be utilized to evaluate com-
munication within human neuronal networks.
HiPSC-NC becomes electrically active within a few
days of differentiation,122 and with new methods like
“patch-seq,” it is possible to study simultaneously
functional (patch clamp) and genetic (ie, single-cell
RNA-seq) characteristics of single cells of a human
neuronal network in vitro.123,124
Another advantage of hiPSC-CM and hiPSC-NC
is the ability to measure cell toxicity of anesthetic
agents. Propofol infusion syndrome is a life-threat-
ening complication of long-term sedation with rather
high doses of propofol.125 Nevertheless, the mecha-
nism of cell toxicity was unclear on the cellular level.
By treating hiPSC-CM with propofol (0–50 µg/mL)
for 48 hours, Kido et al126 revealed that high propofol
concentrations cause mitochondrial dysfunction by
downregulating of peroxisome proliferator-activated
receptor-𝛾-coactivator 1-α (PGC-1α) in human tis-
sue. While the majority of neurotoxicity studies are
based on nonhuman tissue, hiPSC-NC also offered an
opportunity to study the impact of prolonged isoflu-
rane exposure on neuronal survival and neurogenesis
in human tissue.127 Interestingly, the anesthetic toxic-
ity seems to be strongly dependent on cytosolic Ca2+
concentration within the hiPSC-NC.127
The molecular mechanism of some anesthetic actions
are still unknown. Ketamine’s recent renaissance as anti-
depressant agent raised questions about the underlying
mechanism of action: Collo et al128 differentiated hiPSCs
into floor plate-derived midbrain dopaminergic neu-
rons and demonstrated that the structural plasticity and
expression of α-amino-3-hydroxy-5-methyl-4-isoxazole
propionate receptor (AMPAR) and their subunit GluR1
and GluR2 might be causative for positive, antidepres-
sant effects of the drug.
While anesthetic mechanisms have been studied
in nonhuman tissue samples or in large clinical tri-
als, these examples show that with the new hiPSC
 Basic Research Techniques in Anesthesiology
technology a unique venue became available to study
functional and toxicological effects of modern and
future anesthetics in human tissues. By using patient-
specific iPSC-derived tissues, personalized medica-
tions and therapeutic plans can be developed and
transferred into clinical practice.
FUTURE DIRECTIONS OF iPSCs
While iPSC technology has facilitated the develop-
ment of new agents in cardiovascular medicine,129,130
neurology,131 and oncology,132 the use for development
of new perioperative medications has been limited.
Although anesthesia-related drugs have not changed
dramatically over the past 50 years, the fascinating
developments in molecular and cell-based techniques
will pave the way toward improved anesthesia drugs
focusing precisely on the individual patient. Not only
will iPSC technologies allow the development of new
compounds but also facilitate our understanding of
current anesthesia medications. With the rapid prog-
ress in biochip technology, we will hopefully advance
to generating testing platforms which will shorten the
time to determine patient-specific personalized drug
cocktails during the perioperative care period.
CONCLUSIONS
Basic research techniques are rapidly evolving and
increasing in complexity and sophistication. Although
anesthesia is considered safe, we should not ignore
the opportunity to utilize such approaches to study
diseases of relevance to our specialty, to increase our
understanding on how anesthetic drugs function, and
where possible, to move forward drug development
opportunities and personalized medicine. This review
will hopefully encourage others to describe their
sophisticated research tools to (1) make them more
understandable and relevant to the clinically practic-
ing anesthesiologist and (2) to increase the interest and
facilitate the use of these exciting novel techniques to
improve anesthesia care in the future. E
DISCLOSURES
Name: Detlef Obal, MD, PhD, DESA.
Contribution: This author helped review the literature, design
the figures, write the chapter on inducible pluripotent stem cell
technology, design the concept of the review, and review the
manuscript before submission.
Name: Shaogen Wu, MD, PhD.
Contribution: This author helped review the literature, design
the figures, write the chapter on next-generation sequencing,
and review the manuscript before submission.
Name: Andrew McKinstry-Wu, MD.
Contribution: This author helped review the literature, design
the figures, write the chapter on clustered regularly interspaced
short palindromic repeat technology, and review the manu-
script before submission.
Name: Vivianne L. Tawfik, MD, PhD.
Contribution: This author helped review the literature, design
the figures, write the chapter on next-generation sequencing,
460   
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Copyright © 2020 International Anesthesia Research Society. Unauthorized reproduction of this article is prohibited.
conceptualize the manuscript, and review the manuscript
before submission.
This manuscript was handled by: Jean-Francois Pittet, MD.
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