ANALYSIS

Human gene essentiality

István Bartha1, Julia di Iulio1, J. Craig Venter1,2 and Amalio Telenti1,2
Abstract | A gene can be defined as essential when loss of its function compromises viability of the
individual (for example, embryonic lethality) or results in profound loss of fitness. At the
population level, identification of essential genes is accomplished by observing intolerance to
loss‑of‑function variants. Several computational methods are available to score gene essentiality,
and recent progress has been made in defining essentiality in the non-coding genome.
Haploinsufficiency is emerging as a critical aspect of gene essentiality: approximately 3,000
human genes cannot tolerate loss of one of the two alleles. Genes identified as essential in human
cell lines or knockout mice may be distinct from those in living humans. Reconciling these
discrepancies in how we evaluate gene essentiality has applications in clinical genetics and may
offer insights for drug development.

Minimal genome
A genome limited to the
essential genes for life.
Robustness
The ability of a biological
system to keep its behaviour
unchanged under perturbation.
Redundancy
The possibility of having a
function encoded by more
than one gene.
Evolvability
The degree to which an
organism can generate
adaptive solutions to future
environments through
heritable phenotypic variation.
Exome
The subset of the genome that
is part of mature RNAs and
translated into proteins.
1
Human Longevity Inc.,
San Diego,
California 92121, USA.
2
J. Craig Venter Institute,
Capricorn Lane, La Jolla,
California 92037, USA.
Correspondence to
J.C.V. and A.T.
jcventer@jcvi.org;
atelenti@jcvi.org
doi:10.1038/nrg.2017.75
Published online 30 Oct 2017
Gene essentiality emerged as a concept associated with
the notion of the minimal genome1–3. Systematic deletions
of one or more genes in prokaryotic or simple eukary-
otic experimental models have been generated, scored
for viability and fitness, and assessed for mechanisms of
robustness, redundancy and evolvability4. Progress in tech-
nologies for editing and silencing genes extended the
work to more complex models, such as worms and mice.
These studies prompted the identification of essential
components that are shared across different forms of life5.
Human gene essentiality was first associated with
the study of Mendelian diseases, which generally
reflect the consequences of severe genetic lesions on
human fitness. More recently, CRISPR–Cas9 genome-
wide screens have targeted every human gene in tumour
cell lines. These assays measure the consequences of
gene disruption in cell viability assays. However, this
information does not necessarily translate to gene essen-
tiality in vivo, which may be assessed through the collec-
tion of genome sequencing data at the population level.
Therefore, this Analysis article has a strong in vivo organ-
ismal focus supported by advances in genome sequencing
technologies. In large-scale human genome sequencing
projects, essential genes will be those that are rarely or
never disrupted or truncated in the general population.
Here, we review new metrics applied to large exome
and genome sequence data sets for the purpose of
identifying essential genes. Insights from these met-
rics reveal the extent of predicted protein truncation and
other loss‑of‑function variants in the human population.
Importantly, most human loss‑of‑function variants
are rare. Thus, although currently available data are
too sparse for identifying combinations of rare alleles,
there is considerable statistical power when analysing
them in a heterozygous state. Rare loss‑of‑function
variants may exert their influence through a mecha-
nism of haploinsufficiency. Haploinsufficiency is defined
as a dominant phenotype in diploid organisms that are
heterozygous for a loss‑of‑function allele6. In the case
of putative protein-truncating variants such as stop-
gain variants, expression data convincingly demonstrate
that loss of one allele is not remediated by dosage com-
pensation by the intact allele7–12 and thus can result in
haplo­insufficiency. Human disease studies have iden-
tified several hundred haploinsufficient genes13. At the
same time, sequencing projects with large populations
have also identified common loss‑of‑function variants
occurring in a homozygous null state in individuals
who appear healthy. Thus, it is becoming increasingly
possible to define both an ‘essential genome’ (which
includes genes that do not tolerate loss of function) and
a ‘dispensable genome’ (which includes genes that can
be observed with biallelic inactivation in the general
population). This paper reviews the phenotypic con-
sequences of both rare (heterozygous) and common
(homozygous) loss‑of‑function variants.
We also present similarities and differences among
the genetic requirements for a cell to be viable, for via-
ble progeny in the mouse knockout models, and for a
human to reach adult life. Thus, a goal of this article
is to analyse and make sense of these disparate data
sets obtained using conceptually and technically dif-
ferent approaches. As a corollary, this article presents
the extension of the concept of essentiality to the non-­
coding genome. The identification of the most essen-
tial elements of the genome in the human population
provides new insights into human diversity and also
has practical medical applications for disease genetics
and the clinical interpretation of disease-associated
genetic variants.

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A N A LY S I S

Protein truncation
A truncated, incomplete and
usually nonfunctional protein
product. Generally, the result
of stop-gain, frameshift or
splice-donor genetic variants.
Loss‑of‑function variants
Genetic variants that severely
disrupt the function of a
protein. These can be missense
(a change of the codon
resulting in a change in the
amino acid) or nonsense and
protein-truncating variants.
Haploinsufficiency
In a diploid organism, having
only a single functional copy of
a gene (with the other copy
inactivated by mutation), which
is insufficient to maintain
proper gene function.
Stop-gain variants
Also known as nonsense
variants, changes in the genetic
material that result in
premature termination of the
translated protein.

Saturate
When referring to the
generation of gene variants
genome-wide, the sample size
at which all positions in the
genome are seen variant at
least once.
Frameshift variants
Deletions or insertions in the
protein-coding region, the
lengths of which are not
divisible by three, thus
disrupting the reading frame of
the gene.
Synonymous variants
A change of nucleotide that
does not lead to changes in the
amino-acid sequence of a
protein.
Neutral variation
Genetic variants that are not
subjects of natural selection.
ROC curve
(Receiver operating
characteristic curve). A visual
and quantitative method of
evaluating the performance of
binary classifiers. The true
positive rate of a classifier is
plotted against the
false-positive rate.
Metrics of gene essentiality
There are now enough sequenced human exomes and
genomes to saturate certain functional elements (for
example, exons) or mutational classes (for example,
protein-­truncating variants)14–16. Genomic regions or
genes that tolerate variation may carry a high number
of variants, whereas genes that are intolerant to vari-
ation will show a relative depletion. Individuals with
loss‑of‑function variants in essential genes may not be
represented in healthy adult cohorts.
Various scores are proposed to quantify the tol-
erance of a gene to loss‑of‑function variants using
in vivo population-level data of human genetic varia-
tion (TABLE 1). We compare the scores and discuss their
differences while providing guidance for their practical
usage. It should be highlighted that the stated goal of
some of the metrics is the identification of damag-
ing variants and is not explicitly the characterization
of gene essentiality. The available scores differ in the
assumptions, implementation and underlying data
used in building the essentiality metric. An outstand-
ing challenge in all essentiality metrics is in the cali-
bration of an appropriate baseline expectation for the
number of variants in a gene, which will depend on
the length of the coding region, the local nucleotide
context and mutation rates. Due to the more com-
plete understanding of the coding genome and the
better availability of human exome sequencing data,
most of the scores are focused on missense or protein-­
truncating variants, including frameshift variants, early
stop gains and splice-site variants. Synonymous variants
are generally considered biologically silent and thus
provide a natural way to estimate the amount of neu‑
tral variation that is expected in each population for a
given genomic region. However, it should be kept in
mind that synony­mous variants in the protein-coding
region might be under slight evolutionary constraint 17.
Synonymous variants may affect the function of the
translated protein through diverse cellular mecha-
nisms, including control of transcription rate, RNA
structure and protein folding efficiency 18.
The main principle of all scores is to rank genes by
the footprint of negative selection acting on the genes
against protein-truncating variation. Petrovski’s ‘resid-
ual variation intolerance score’ (RVIS)19 and Rackham’s
EvoTol20 relate the amount of common loss‑of‑­function
variation to that of the total gene variation. Other
scores are based on the original work of Samocha et al.
(Missense Z‑score)21, which sets up a baseline expec­
tation of mutation count per gene based on the sequence
context, local mutation rate, sequencing depth and, most
importantly, sample size. Fadista’s LoFtool22 combines
the neutral mutation rate of Samocha et al.21 and the
evolutionary information in EvoTol20. The baseline neu-
tral expectation is compared with the observed counts
of loss‑of‑function variants in the Missense Z-score21,
in Bartha’s probability of haploinsufficiency (Phi)23
and in Lek’s probability of loss‑of‑function intolerance
(pLI)15. Finally, recent work by Cassa et al.24 describes a
metric (shet) that provides Bayesian estimates of the selec-
tion coefficient against heterozygous loss‑of‑function
variation. The various scores were developed or updated
using the Exome Aggregation Consortium (ExAC)
sample of 60,706 human exomes15. Therefore, the use of
these metrics on the same data set allows the assessment
of agreement. Indeed, these scores are highly correlated
with one another (FIG. 1). The highest correlation arises
between the two scores (Phi and pLI) that use Poisson
mixture models in their approaches (Supplementary
information S1 (box)). The key differential character-
istics of the scores are presented in TABLE 1. All scores’
URLs and values are included in Supplementary
information S2 (table).
The performance of the scores is assessed by predict-
ing variants causing Mendelian disorders (TABLE 1) and
by demonstrating their use for variant prioritization in
the clinical genetic setting. It is, however, difficult to
draw a uniform picture from the original publications,
as each tool was benchmarked against different truth
sets and trained on different genetic data. For example,
shet is capable of predicting the inheritance mode of a
loss‑of‑­function variant with >85% accuracy and sensi-
tivity, and most methods have an area under the receiver
operating characteristic curve (ROC curve) of 70–90%
against Online Mendelian Inheritance in Man (OMIM)
haploinsufficient genes. No matter how they are imple-
mented, all these methods are inherently dependent on
the gene length, as it is difficult to assert the depletion
of observed variants against an already low expectation of
the number of variants in a short gene. Short gene length
will lead to false-negative calls (that is, genes erroneously
being called as non-essential), but it will not lead to
false-­positive calls of haploinsufficiency.
Another important consideration is that these tools
estimate essentiality as a continuum of values but are
frequently used as dichotomized scores. TABLE 1 indi-
cates the distribution of each of the scores and the cut-off
(if any) that was used by their authors to evaluate patho-
genicity. The broadly used cut-off of pLI >0.9, proposed
by Lek et al.15, results in 3,230 genes that can be consid-
ered highly essential. Here, for the purpose of comparing
the various scores and for comparisons between human
in vivo data, in vitro tumour cell data and mouse knock-
out data, we display ranked continuous scores or use a
cut-off of the 85th percentile, which approximates the
commonly used pLI >0.9.
We highlight essentiality scores that are primarily
grounded on human population genetic information
explored using unsupervised analyses. There are also
supervised predictors of haploinsufficiency that integrate
genomic features, including functional annotation, met-
rics of network topology, evolutionary and intraspecies
population-level data. Representative works are those
of Dang et al.25, Khurana et al.26, Steinberg et al.27 and
Shihab et al.28. However, it should be noted that Shihab
et al.28 found that the most informative feature was the
population-level data captured by the Missense Z-score21.
Characteristics of essential genes
The use of human population data to identify genes
that appear to be essential in vivo invites the com-
parison to essential genes described in other settings.

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Table 1 | Metrics of gene essentiality for human population sequence data

Score Residual EvoTol Missense Probability Probability of LoFtool Selective effects
variation Z‑score of haplo­ loss‑of‑function for hetero­
intolerance insufficiency intolerance (pLI) zygous protein-
score (RVIS) (Phi) truncating
variants (shet)
Sample size 6,503 exomes 1 million variants 1,000 Genomes 10,500 exomes 60,706 exomes 60,706 exomes 60,706 exomes
(ESP), updated to from dbSNP Project data (ESP + UK10K + from ExAC from ExAC from ExAC
60,706 on their to calculate  CoLaus),
website the baseline autosomes
mutational only, updated
rate of de novo for 60,706
mutation; ESP exomes
for the observed
variant counts
Method Residuals derived Residuals derived Z‑Score to Posterior Posterior Heuristic, builds Bayesian
from the linear from the linear quantify the probabilities probabilities from on EvoTol20 estimation of
regression of regression of difference from a a three-state and mutation the selection
the number the number between the two-state Poisson mixture model from coefficient on
of common of common observed Poisson mixture model the Missense heterozygous loss
functional functional missense model Z-score21 of function
variants on the variants on the variants and
total number of total number of the expectation
variants variants based on a
mutation model
Distinctive Introduces Combines Uses a neutral Uses a mixture Unprecedented Non-parametric Direct estimation
feature the concept of intraspecies and mutation model model to sample size combination of the selection
applying human interspecies as a baseline estimate haplo­ of mutation acting on the gene
population information in a insufficiency rates, functional
genetics to similar framework predictions and
assess function, to RVIS variation data
pathogenicity and
essentiality
AF filter >0.1% NA Singleton <1% <0.1% NA <0.1%
Variant class SNVs only; SNVs only; Missense Stop-gain or Stop-gain, Stop-gain, Stop-gain, splice
missense, considered to frameshift splice, frameshift splice, (VEP-LOFTEE)
stop-gain, be functional (SnpEff) (VEP-LOFTEE) frameshift
stop-lost, based on fathmm (VEP-LOFTEE)
prediction of prediction
splice-site effects
as provided by ESP
Reported 58% against ROC shown, no 87% against 76% against Not reported 86% against OMIM not
precision OMIM disease AUC reported, de novo OMIM haplo‑­ de novo reported, 93%
against OMIM genes, 78% outperforms RVIS OMIM haplo‑­ insufficient OMIM haplo-­ to identify
genes (ROC/ against OMIM insufficient genes insufficient inheritance mode
AUC) haploinsufficient genes genes on 459 genes
genes or carrying involved in clinical
de novo variants, disease
80% against
de novo OMIM
haploinsufficient
genes
Functional High fraction of Nuclear Higher Z‑score Fewer Highly expressed Enriched in Developmental
and clinical developmental receptors and in genes paralogues; genes; depleted genes expressed pathways and
correlates genes in the top metabolic genes with de novo more central in in eQTL hits; preferentially in transcriptional
quartile are intolerant loss‑of‑function protein–protein enriched in the brain regulators are
to predicted mutations in interaction GWAS hits; more enriched in high
damaging autism spectrum networks; protein–protein shet values. Positive
variants disorders or enrichment interactions; correlation with
intellectual in genes with gene-set protein–protein
disability cases lethal mouse enrichment interaction count
knockout analysis results
phenotype, (spliceosome,
more likely to ribosome and
be in protein proteasome); 50%
complexes; of assessed human
enriched in orthologues of
genes for which mouse genes
loss of function with conditional
leads to loss of lethal knockout
cell viability; phenotype are
more conserved essential

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Table 1 (cont.) | Metrics of gene essentiality for human population sequence data
Score Residual EvoTol Missense Probability Probability of LoFtool Selective effects
variation Z‑score of haplo­ loss‑of‑function for hetero­
intolerance insufficiency intolerance (pLI) zygous protein-
score (RVIS) (Phi) truncating
variants (shet)
Interpretation Lower Lower Higher Higher Higher Lower value =  Higher
value = more value = more value = more value = more value = more more intolerant value = more
intolerant intolerant intolerant intolerant intolerant intolerant
Thresholds for Standard normal; Percentiles; the Standard normal Bimodal on Bimodal on [0,1]; Percentile; the Inverse Gaussian
pathogenicity the authors authors evaluate [0,1]; the the authors authors evaluate
evaluate the the lowest authors evaluate the set the lowest
lowest quartile quartile evaluate the set >0.9 quartile
>0.95
Refs 19 20 21 23 15 22 24
AUC, area under the curve; AF, allele frequency; CoLaus, Cohort Study of Lausanne; dbSNP, The Single Nucleotide Polymorphism database; eQTL, expression
quantitative trait locus; ESP, Exome Sequencing Project; ExAC, Exome Aggregation Consortium; fathmm, functional analysis through hidden Markov models;
GWAS, genome-wide association study; NA, not applicable; OMIM, Online Mendelian Inheritance in Man; ROC: receiver operating characteristic; SNV,
single-nucleotide variant; UK10K, the United Kingdom 10,000 Genomes Project; VEP-LOFTEE, Loss‑Of‑Function Transcript Effect Estimator plugin for the Ensembl
Variant Effect Predictor (VEP).

Expression quantitative
trait loci
(eQTLs). Loci where variation is
associated with differential
expression of a gene.
Haploid
Of cells, containing a single set
of chromosomes.
Indeed, multiple systems have been developed to iden-
tify essential genes in prokaryotic or eukaryotic mod-
els, in simple or complex forms of life, and in in vivo or
in vitro settings. The Database of Essential Genes (DEG)5
compiles in version 14.7 a total of 53,394 essential genes
and 786 essential non-coding sequences among bac­
teria (18,285 essential elements), archaea (519 essential
elements) and eukaryotes (34,590 essential elements).
Pioneering studies in yeast allowed the systematic
deletion of all genes individually. Giaver et al.29 estimated
that approximately 20% of the genes were required for via-
bility. These encode core components of the cell, such as
proteins involved in transcription and metabolism. Those
essential components are largely shared with other eukar-
yotic forms of life, such as Caenorhabditis elegans. RNA
interference (RNAi) screens in the worm indicate that
>50% of worm essential genes are orthologous to yeast
essential genes30. All the studies, whether in yeast, worms,
mice or, more specifically, in humans, describe essential
genes as located in critical places in the core develop­
mental, metabolic and signalling pathways. Specifically,
compared with other genes, essential genes are described
as having more protein–protein interaction partners,
being more centrally located in protein–protein networks,
being broadly and strongly expressed and depleted in
expression quantitative trait loci (eQTLs), having higher
protein abundance, being more conserved between spe-
cies, having significantly fewer paralogous genes in a
genome, and being associated with developmental path-
ways and embryonic lethality 23,24,31–35. In particular, the
role of essential genes in protein–protein interaction net-
works36 has received considerable experimental support37.
Proteins are classified as ‘indispensable’ based on their
impact on the network upon removal of the specific pro-
tein. Recent analysis of the interaction network of 6,339
human proteins and 34,813 interactions identified 21% of
the proteins in the network as indispensable33. The in­­
dispensable proteins often harbour disease-causing var-
Below, we emphasize the correspondence between
in vitro screens in human cell lines, mouse models,
and in vivo human genetic population data. The reader
needs to consider the profound experimental differ-
ences of what is tested for, as well as the implications
for what is deemed to be essential across these disparate
approaches (TABLE 2).
Human cell lines. There have been several CRISPR–
Cas9 studies38–40 that targeted every gene in human cell
lines and scored for cell viability. Overall, these in vitro
screens identified components of fundamental path-
ways that are expressed at high levels. They also identi-
fied enrichment for genes involved in RNA processing.
Work by several groups, and encompassing many cell
lines, showed a high degree of overlap in the pattern of
enrichment but also revealed differences specific to each
cell line that may reflect the developmental origin, onco-
genic drivers, paralogous gene expression patterns and
chromosomal structure of each cell line40. One impor-
tant caveat of cell-based assays for gene essentiality is
that they address the biology of tumour cell lines and
generally test for complete knockouts. FIGURE 1 illustrates
the correlation between essential genes identified in cell-
based assays and those identified by in vivo genetic stud-
ies using the scores presented in the previous section. It
is apparent from this analysis that assays on tumour cells
largely agree on the sets of genes considered essential
in vitro, in particular for a given laboratory. However,
the cell-based results do not share the same core set of
essential genes (FIG. 2) with those identified in vivo in
human population studies.
The observed differences between in vitro screens
and in vivo population studies may reflect different
requirements for tumour cell versus organismal viability.
They may also reflect ploidy differences because some
of the investigated tumour cell lines are fully or partially
haploid (KBM7), where a single CRISPR–Cas9 deletion

Ploidy
iants and are targets of drugs, which made the authors results in a knockout, whereas other cell lines are dip-
The number of sets of state that altering the network properties is critical for the loid or near-diploid (Raji, Jiyoye, HCT116 and RPE1),
chromosomes in a cell. transition between healthy and disease states33. hypotriploid (K562), or of greater ploidy complexity

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 A N A LY S I S

Hart DLD1 loss‑of‑function variants in essential genes may also

Hart GBM be lethal in mice34,44,45. By contrast, human population
Hart RPE1 analyses predominantly score on loss of one allele or
Hart HeLa on depletion of variants in a gene. This implies that
Hart HCT116 human carriers may have severe phenotypes that lead to
Cell viability data exclusion from adult cohorts such as ExAC.
Blomen KBM7
Wang Raji Georgi et al.34 characterized the patterns of genetic
Wang Jiyoye variation of 2,472 human orthologues of known essen-
Wang K562 tial genes in the mouse. Consistent with the action of
Wang KBM7 purifying selection, these genes are characterized by
Phi reduced levels of sequence variation, skewed towards
pLi more rare variants, and increased conservation across
In vivo data s_het the primate and rodent lineages relative to the remainder
missense-Z of genes in the genome. A list of 593 recently reported
LofTool essential genes in mice46 combined with null alleles from
RVIS the Mouse Genome Informatics (MGI) database results
in 3,326 essential genes that have orthologues in the
RVIS
LofTool
missense-Z
s_het
pLi
Phi
Wang KBM7
Wang K562
Wang Jiyoye
Wang Raji
Blomen KBM7
Hart HCT116
Hart HeLa
Hart RPE1
Hart GBM
Hart DLD1
human genome. Comparison of these genes to human
genes ranked by their essentiality scores indicates an
overlap of 932 essential genes (FIG. 2). This core set of
932 genes shows a modest enrichment for functions in
1.0 0 –1.0 transcription, regulation of gene expression and nucleo­
Correlation In vivo data Cell viability data tide metabolism, as well as for autosomal dominant
Figure 1 | Rank correlation of essential gene sets across human population data disease genes.
and cell-based CRISPR–Cas9 screens. The essentiality metrics Nature Reviews
residual | Genetics
variation There are also discordant gene sets between mouse
intolerance score (RVIS) , LoFtool , Missense-Z , shet (REF. 24), probability of
19 22 21 and human data: 2,394 genes are lethal in knockout mice
loss‑of‑function intolerance (pLI)15 and probability of haploinsufficiency (Phi)23 are but are not scored as essential in humans (FIG. 2). The
compared to cell viability data from studies of Wang86, Blomen38 and Hart39. EvoTol20 is considerable size of the discordant sets underlies dif-
not depicted due to the lack of availability of updated scores using the Exome ferences between synthetic experiments and data from
Aggregation Consortium (ExAC) sample. There is a high degree of consistency internal to outbred human populations. For this discordant set, a
the in vitro screens, mostly within a given laboratory. Of note, the human population-­
reason for the discrepancy is also the lack of power of the
genetics-based scores were calculated on the same data source of 60,706 samples from
bioinformatics scores: short genes and recessively acting
ExAC15. See Supplementary information S1 (box) for data sets and code needed to
generate the figure. genes may go undetected. There are also 1,282 genes that
score as essential in the human population. Of these, 495
have been evaluated in mice; some of these genes may
(GBM514, HeLa and DLD1). A gene may also be needed also be considered essential in mice when accounting
in a cell line for the culture conditions tested, whereas for loss of fitness rather than merely a binary viable
an essential gene in vivo could be needed in any impor- versus nonviable classification of essentiality. Another
tant tissue at any stage of development. Finally, the 787 are scored as essential in vivo in humans but have
main signal from human population data derives from not been tested or reported in knockout mice. Because
the identification of haploinsufficiency and, to a lesser of the production process for knockout mice, which
extent, from the contribution of null states due to homo­ until the advent of genome editing included the use
zygous loss‑of‑function and biallelic inactivation. This of a male embryonic stem cell line, the vast majority of
is an important consideration when comparing in vivo haplo­insufficient essential genes would simply be missed
results with those from near-haploid screens in KBM7, (those that are haplolethal) and indistinguishable from a
a cell line that is already, at baseline, in a state of hemi­ technical failure. These considerations notwithstanding,
zygosity and only scoring major effects when genes are there is general agreement regarding the pathways that
fully knocked out. are enriched in or depleted of essential genes in humans
and mice. This supports the notion that the in vivo con-
Knockout mice. The mouse is the most commonly ditions screened in the mouse model can reflect human
used model organism in human disease research41. consequences with respect to loss of function in those
Roughly 30% of genes in the mouse genome may be genes, despite the notable differences between null data
necessary for survival to adulthood42. However, the use- in mice (dominant and recessive) and haploinsufficiency
fulness of mouse models has been questioned for their data in humans (dominant only).
recapitu­lation of human conditions43. It is important to
emphasize what is scored in mice: the impact of bial- The core set of essential genes in mice and humans.
lelic inactivation on embryonic lethality. This implies From the discussion above, it follows that there are
that mice that were crossed in these experiments car- important considerations when comparing essen-
Hemizygosity
ried heterozygous loss‑of‑function genotypes and were tial gene sets defined by screens and metrics that test
The absence of one copy of a viable and fertile, although they might have had a clin- fundamentally different systems. The requirements of
gene in diploid cells. ical or laboratory phenotype. However, heterozygous tumour cells of variable ploidy, the viability of knockout

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Table 2 | Differences between studies of gene essentiality in the human population, human cells and mice
Human adult population Human cell lines Mouse models
Source of data Large-scale population sequencing projects Cancer cell lines (KBM7, Raji, Jiyoye, HCT116, Knockout mice from the MGI
(for example, ExAC) RPE1, K562, GBM514, HeLa and DLD1) database
Test Rare heterozygous loss‑of‑function variants* CRISPR–Cas9‑mediated knockout (haploid, Biallelic inactivation
diploid or complex ploidy cell lines)
Metric Essentiality scores (RVIS, Missense Z‑score, Cellular viability Embryonic lethality
EvoTol, Phi, pLI, LoFtool and shet)
Functional and Haploinsufficiency; dominant effects Dominant and recessive effects Recessive effects
genetic model
Additional Biallelic inactivation due to rare The requirements for viability of the The mice that were crossed
considerations loss‑of‑function variants is rarely scored specific tumour cell depend on the culture in these experiments carried
or observed. Common homozygous conditions tested. An essential gene in vivo heterozygous loss‑of‑function
loss‑of‑function variants are unlikely to be may be required in any important tissue at genotypes but were viable and
associated with fitness consequences. any stage of development. fertile.
*Can be extended to testing of depletion of non-synonymous variants. ExAC, Exome Aggregation Consortium; MGI, Mouse Genome Informatics; Phi, probability of
haploinsufficiency; pLI, probability of loss‑of‑function intolerance; RVIS, residual variation intolerance score.

mice bred from heterozygous animals, and the obser­
vation of heterozygous genotypes associated with haplo­
insufficiency in the human population test profoundly
different biological conditions. There is, however, a
core set of 188 genes that are called essential by all three
orthogonal approaches (FIG. 2). These genes represent
major cellular functions, prominently, splicing, nuclear
transport, nucleosome architecture, mitosis and the cell
cycle (FIG. 3).
Essential genes of unknown function in humans. The
design of the minimal genome of Mycoplasma mycoides
(JCV‑syn1.0) led to the unexpected observation of
149 genes that could not be assigned to a specific bio­
logical function3. Of these, 55 have completely unknown
functions, suggesting the presence of undiscovered func-
tions that are essential for life. Dey et al.47 investigated
the “dark matter of the human protein-coding genome”,
which they estimated as being composed of more than
6,000 poorly studied genes. By profiling the entire
human protein-coding genome across 177 eukaryotic
species by using phylogenetics, they predicted the func-
tions for hundreds of the poorly characterized genes.
As an approximation of the identification of essential
genes of unknown function, we assessed how many of
the most essential genes (in the top 5% of essentiality in
either the human population genetic scores or the cell-
line-based metrics) returned no articles in PubMed.
This approach rendered a list of 19 expressed essential
genes of uncharacterized function. These genes include
PCNX1 (an evolutionarily conserved transmembrane
protein similar to the pecanex protein in Drosophila
melanogaster), TBC1D3C (which belongs to a cluster of
related genes that may be involved in GTPase signal-
ling and vesicle trafficking), POLR3H (an uncharacter-
ized subunit of RNA polymerase III that is conserved
across species), MRPL57 and MRPS14 (proteins that
belong to undetermined ribosomal subunits that seem
specific to animal mitoribosomes), DDX55 (a putative
RNA helicase that may be involved in a range of nuclear
processes including translational initiation, nuclear and
mitochondrial splicing, and ribosome and spliceosome
assembly), CBWD3 and CBWD5 (cobalamin synthase
W-domain-containing proteins) and TRIM51 (two
organisms have orthologues with the human gene).
Other genes in the set are also broadly conserved across
species: BRIX1, TANGO6, EIF5AL1, ZMAT2, ZSWIM8,
DENND4B, TEDC1 (also known as C14orf80), ARMC7,
CCDC84 and RTFDC1.
Two genes in the short list, ARMC7, CCDC84, were
identified as essential in cell lines and were subjected
to dedicated functional analyses39. CCDC84 exhibited
enriched nuclear staining and interacted with different
sets of proteins that are predicted to participate in mRNA
splicing, and it thus may be a component of the PRPF
splicing complex. ARMC7 co‑immuno­precipitated with
the poorly characterized protein RBM48. The investi-
gators indicate that RBM48 contains an RNA-binding
motif, and ARMC7 is an Armadillo-repeat containing
protein, which was interpreted as RBM48–ARMC7
being an essential protein complex with a role in RNA
metabolism and/or transcription. Both genes were
found to be amplified across several cancer tissues and
cell lines39.
As this Analysis article went to press, Cassa et al.
also described a promising set of essential genes that
lack functional or disease associations24. These exer-
cises for identifying essential uncharacterized genes are
not exhaustive and may be confounded by the legacy of
previous gene names or by paralogues. However, these
analyses serve to highlight the opportunities to delve
into the issue of essential genes of unknown function in
the human genome48.
Pathogenic variants in essential genes
In 2012, MacArthur et al.10 applied stringent filters
to 2,951 putative loss‑of‑function variants obtained
from 185 human genomes to determine their true
prevalence and properties. This study concluded that
human genomes typically contain approximately 100
loss‑of‑function variants. Their prediction implies
that up to 20 genes could be completely inactivated
by homozygous loss of function in each individual.
However, genes carrying common loss‑of‑function

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 A N A LY S I S

Essential in human populations

• Whole organism might need functions
not needed for simple cellular systems 1,894
• Enriched in dominant disease genes 1,894
744
744
1,059
485
Essential in knockout mice 188 188
• Enriched in recessive disease genes
223 500 10 500
Subtract genes for
which no knockout 45
Essential in human cell lines mice have been
• Cell lines are not representative of produced
1,098
whole organisms
• Enriched in recessive disease genes

Total: 5,706 Total: 3,866

Figure 2 | Consistency of essentiality calls across human in vivo, mouse in vivo, and CRISPR–Cas9 cell line data sets.
Venn diagrams depicting the 3,326 mouse genes identified as essential by Dickinson et al.46 and theNature Reviews | Mouse
International Genetics
Phenotyping Consortium (yellow); the top 15% human essential genes as defined by RVIS, pLI, Phi, missense Z‑score,
LoFtool and shet from human population exome sequence data (red); and essential genes as identified by CRISPR–Cas9
screens on human cell lines (blue). The total number of genes marked as essential by any of the methods is 5,706
(left panel). Not all genes may have been scored in all three systems, in particular in the mouse models; hence, the right
panel only shows genes for which mouse knockouts have been generated. The nature of the 188 genes in the intersection
(that is, those scoring as essential in all three systems) is depicted in FIG. 3.

Compound heterozygosity
The state in which both alleles
of a gene carry a (deleterious)
variant, but those variants are
different.
variants were observed to be less central to key cellu-
lar pathways and to be strongly enriched for functional
categories, such as olfactory reception, that are not
critical for fitness and survival. At the other extreme
of the spectrum, they observed heterozygous carriers of
rare variants for numerous recessive disease genes. As
described above, the current scale of genome and exome
data, as well as the new metrics for essentiality, support
a reassessment of the disease consequences of variants
in essential genes.
The analysis by Cassa et al.24 of heterozygous protein-­
truncating variants in over 60,000 ExAC individuals
used the shet score. The highest shet values predict pheno-
typic severity, age of onset and penetrance for Mendelian
disease-associated genes. In addition, genes involved in
neurological phenotypes, including autism, congenital
heart disease and inherited cancer risk, seem to be under
more intense selection, that is, more essential. Overall,
quantitative estimates of essentiality appear particularly
useful in Mendelian disease gene discovery efforts. There
is emerging data suggesting that loss‑of‑function var-
iants are more frequently observed in younger people,
supporting the notion that they may limit healthspan49.
Among over 10,000 deeply sequenced genomes at
Human Longevity Inc14., we have observed 505,906 puta-
tive loss‑of‑function variants, representing an average
of 40.7 per individual genome. In addition, there are,
on average, 0.6 pathogenic or likely pathogenic ClinVar
variants and 3.9 high-confidence disease-causing muta-
tions in the Human Gene Mutation Database (HGMD)
per individual. Essential genes are enriched for patho­
genic variants that are annotated in human genetic
variant databases (ClinVar and HGMD)50,51 (FIG. 4). The
enrichment is magnified by the parallel depletion of rare
missense variants in those genes (FIG. 4). The stable rates
of synonymous variation across various levels of gene
essentiality are consistent with the neutral model, where
synonymous variants are randomly distributed across
the spectrum of essentiality. It is also of note that when
the same analysis is done with data from CRISPR–Cas9
screens, the depletion of rare variants occurs only at the
highest scores of in vitro loss of viability. This result can
be interpreted as an indication that only the most severe
in vitro cellular phenotypes correlate with the in vivo
human population data. This is consistent with the
study design: of all the genes for which biallelic loss‑of‑­
function variants impair viability based on in vitro
screens, the mildest effects are expected to be recessive
(no noticeable effect when heterozygous), whereas the
most severe could be expected to be haplo­insufficient
(organismal impairment when hetero­zygous, enabling
their discovery by the human population sequencing
approaches). Overall, there are 395 diseases that are
associated with genes that are essential in the human
population and 681 diseases associated with genes that
are essential in mouse models or cell line CRISPR–Cas9
screens. The list of diseases associated with essen-
tial genes is included in Supplementary information 
S3 (table).
Consequences of biallelic inactivation
As discussed, most of the signal of essentiality in humans
is derived from the observation of rare heterozygous
loss‑of‑function variants. However, there are also
common (high allele frequency) loss‑of‑function var-
iants that effectively result in null individuals through
homozygosity or compound heterozygosity. Loss of those
genes is likely to have weak or neutral effects on fitness
and health because they are observed in presumably
healthy adults10,52–54. ExAC, which comprises data from

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A N A LY S I S

WDR1

TLN1
PDPK1
MAPKAP1 ATP5A1
SOS1 PPRC1 ATP5B
RICTOR
HSPA5 ZBTB17 BPTF GABPA
MTOR
MYH9 NRF1
RPTOR HSPD1 NCKAP1
VPS35
ACTG1 ACTR3 HMGCR
EIF3B SP1 SMARCA5 NRBP1
INO80 BRD4
PTPN11 SMG1 RUVBL2 SAP130
TRRAP MED12
UPF1 YY1 ACTL6A DNMT1
UPF2 RAF1 CCT3 CHAF1A
MED1
CASC3 TRIM28
ACTB SRF
PPP2CA
SHOC2
INTS2 SIN3A
SMARCA4
PPP1CB SMARCB1
PPP2R1A CHD4
INTS6
NPLOC4 PRMT5 GTF2A1
VCP
COPS5 COPS2
CUL1 GPS1
SFPQ
CNOT9 CUL3 PSMC3 POLR1B

KIF11 CDK1 WEE1 DDB1

DYNC1H1 PKM
EXOC8 ANAPC2 G6PD
PLK1 CKAP5 PRMT1
DCTN1 ESPL1 WAPL POLR2A
PAFAH1B1 CTCF DGCR8
SMC3 PDS5A DROSHA
LIN9 CTR9 CDC73
NUP85
RAD21 SRSF3 TOP1 RTF1 MEN1
NUP155 HNRNPU
NIPBL SSRP1 SETD1A
RANBP2 SRSF1
TPX2 HNRNPL SF3B1
KPNB1 RAE1 SRSF7 SF3A1 XAB2 KMT2D
NUP98 SNRNP200 DHX9
ILF3 PRPF19 RBM39
EEF2
PRPF3 Cell cycle arrest
and/or DNA repair
RAD51 GTF3C1 XRCC5 Microtubule and/or mitosis
AP2M1 SYMPK DICER1
UBR4 Nuclear transport
DDX5 SRRT Other
COPG1 RBBP6 REV3L SF1 AGO2
Splicing
Transcription,
nucleosome, epigenetics

Figure 3 | Core set of essential genes in mice and humans. A total of 188 genes are scored as essential
NatureinReviews
human population
| Genetics
studies, human cell-line CRISPR–Cas9 screens and knockout mice. These genes represent major cellular functions (inset).
Genes that are part of Gene Ontology gene sets that are significantly enriched in the core essential genes are colour coded
according to major cellular functions. The figure is built using STRING DB experimentally validated and database-derived
interactions. See Supplementary information S1 (box) for the data sets and code needed to generate the figure.

predominantly outbred populations, identified 1,775
genes with predicted biallelic loss of function in 60,706
individuals15. In the Icelandic population studies, 1,171
genes were predicted to be null among 104,220 indi-
viduals55. A study of 3,222 British adults of Pakistani
heritage with high parental relatedness identified 1,111
homozygous genotypes with predicted loss of function
in 781 genes54. Although the latter study focused on
rare variants, it did not record adverse consequences
of the loss of function, as assessed by rates of medical
consultation and drug prescription54. The population
bottleneck of the Finnish founder population facilitates
the study of low-frequency loss‑of‑function variants
as well as Mendelian diseases. A study of 3,000 Finns
described a significant enrichment of low-frequency
(0.5–5%) loss‑of‑function variants56. Simulation based
on these data suggests that although deleterious vari-
ants are inevitably pushed to extremely low frequency
after 1,000 or more generations, they can easily persist
at frequencies between 0.1 and 1% up to 100 generations
after a bottleneck56. The latest published study of 10,503
adult participants in the Pakistan Risk of Myocardial
Infarction Study 57 identified 49,138 rare loss‑of‑­function
variants estimated to knock out 1,317 genes, each in at

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 A N A LY S I S

Synonymous Missense Pathogenic (n = 273). This includes eight genes (HSPD1, DNM2,
CASC3, ABCB7, NUP85, LIN9, BPTF and UBR4) that
Synonymous or missense variants per kb [10K individuals]

40 In vivo 6 are deemed to be essential by all three systems and mod-

els. However, there is evidence that the observed homo­

Pathogenic variants per kb [ClinVar and HGMD]

zygous variants in these eight genes do not contribute to
actual loss of protein function: variants in those genes are
35
5 positioned in the last exon or in non-conserved exons or
may represent sequencing errors.
In vitro It is thus important to exclude truncating variants of
30 limited functional impact. The assessment of putative
4 loss‑of‑function variants, whether rare or common,
requires attention to the percentage of sequence affected,
25 loss of functional domains, proportion of isoforms
affected, principal isoform damage, and degra­dation
3 of the transcript by nonsense-mediated mRNA decay
(NMD)8,11,58,59. Specifically, there is depletion of com-
20
mon loss‑of‑function variants in principal isoforms and
In vivo
in regions more than 50 nucleotides upstream of the last
In vitro 2 exon–exon junction8. Other putative loss‑of‑function
0.00 0.25 0.50 0.75 1.00
variants that may be excluded are those in non-­canonical
splice sites or exons flanked by non-canonical splice
Low High
Essentiality sites, splice-site mutations at small (<15 bp) introns,
and where the purported loss‑of‑function allele is
Figure 4 | Pathogenic variants in essential genes. For the left axis, the plot uses variants
Nature Reviews | Genetics observed across primates57. The program LOFTEE
from over 10,000 deep-sequenced genomes9. Depicted are the distributions of rare (allele
frequency <0.001) synonymous (grey; N = 494,641) and missense (red; N = 887,534) variants
(Loss‑Of‑Function Transcript Effect Estimator), which is
throughout the essentiality spectrum. Red and grey solid lines are computed with a plugin for the Ensembl Variant Effect Predictor (VEP)
essentiality scores using human population data; dotted lines are computed with the implements these practices. Finally, manual curation
essentiality scores obtained with CRISPR–Cas9 screens in vitro. The right axis depicts the may be required to exclude variants with few or poorly
distribution of pathogenic variants (purple; N = 113,951) obtained from public databases supported sequencing reads, and for any given putative
(ClinVar and the Human Gene Mutation Database (HGMD)). It shows a significant loss‑of‑function variant, experimental validation will be
enrichment in pathogenic variants in essential genes. The more essential a gene is, the less required to prove loss‑of‑function57. In summary, just as
likely it is to tolerate missense variation but the more likely that this variation results in there is a case for an essential genome of around 3,000
pathogenicity. By contrast, the distribution of rare synonymous variants (grey) across the genes, there is also substantial evidence to support a case
essentiality spectrum is flat, which is consistent with a neutral model, that is, the absence
for a 3,000‑gene or larger dispensable genome.
of purifying selection.
Essentiality of the non-coding genome
There is increasing awareness that genetic variants,
least one participant. The study explored in detail the including single-nucleotide and structural and copy
health consequences of null phenotypes of PLA2G7, number variants, in the non-coding regions of the human
CYP2F1, TREH, A3GALT2, NRG4, SLC9A3R1, and genome can play an important role in human traits and
APOC3 (REF. 57). These studies are converging on a diseases. In fact, most of the genome-wide association
catalogue of human loss‑of‑function variants. Projects study (GWAS) variants map to non-coding regions
such as The Broad Institute’s Human Knockout Project (reviewed in REF. 60), and there are increasing numbers
will leverage previously generated and new functional of reports of Mendelian disorders that map outside the
genomic data to validate a subset of these variants. It is protein-coding genome61–64. Understanding the relation-
expected that many instances of biallelic inactivation will ship between essentiality, genetic constraint of regulatory
require genotype-based recall of the individuals for deep elements, and pathogenicity would be important for
pheno­typing to gauge the functional consequences53. progress in understanding the non-coding genome.
We analysed the nature of genes that tolerate a puta- Can the idea that natural variation saturates the
tive null state due to homozygous loss‑of‑function var- genome at non-essential regions extend to non-­protein-
iants in the adult human population. There are 3,296 coding and regulatory regions? There are two ortho­gonal
genes in ExAC with at least one homozygous features of coding regions that are not available in the
loss‑of‑function variant. The median allele frequency is non-coding genome. First, the non-coding genome lacks
greater than 1 per 1,000. As expected, this set of genes strong landmark features, which could serve as a unit
Nonsense-mediated mRNA is enriched for drug metabolism genes and olfactory similar to an exon. Second, certain mutational classes
decay receptors and scored for low essentiality. These data sup- in the coding genome (for example, protein-truncating
(NMD). A cellular pathway that port the concept of a ‘dispensable genome’ as profiled variants) are easier to score than those in the non-­coding
serves to recognize and by the various publications summarized in the previous genome. However, recent advances in understanding
degrade mRNAs with
translation termination codons
para­graph. However, there is some overlap with genes that conservation65 and CRISPR–Cas9 screening 66 of the
that are positioned in are scored as essential in the human population (n = 117), non-coding genome supports extending the concept of
abnormal contexts. in CRISPR–Cas9 cell lines (n = 159) and in knockout mice essentiality beyond the human exome.

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The functional characterization of the non-­coding
genome has been guided prominently by bio­chemical
data as represented by the Encyclopedia of DNA
Elements (ENCODE) project 67 and by scores that
serve to predict the pathogenicity of genetic variants.
Prominent among these scores are CADD68, DeepSEA69
and Eigen 70. CADD, the Combined Annotation-
Dependent Depletion score, uses evolutionary infor­
mation (human–chimpanzee fixed or nearly fixed recent
evolutionary changes), annotation from Ensembl VEP,
data from the ENCODE project and information from
UCSC genome browser tracks (sequence conservation
scores from GERP, phastCons and phyloP; functional
genomics data such as DNase hypersensitivity and tran-
scription factor binding; transcript information includ-
ing distance to exon–intron boundaries or expression
levels in commonly studied cell lines; and protein-level
scores such as Grantham, SIFT and PolyPhen) 68.
DeepSEA69 was developed to directly learn a regulatory
sequence code from large-scale chromatin-profiling
data. DeepSEA enables the prediction of the chromatin
effects of sequence alterations with single-nucleotide
sensitivity to prioritize functional variants including
eQTLs and disease-associated variants69. More recently,
Eigen was developed as an unsupervised approach for
integrating these different annotations into one meas-
ure of functional importance70. Eigen is dependent on
annotation but independent of any labelled training
data. There are additional computational methods to
prioritize non-coding variants with functional effects
(reviewed in REF. 71). Overall, these scores support var-
iant prioritization for pathogenicity by using extensive
evolutionary, biochemical and functional data.
More recently, the sequence context has been used to
study polymorphisms at the genome level. Specifically,
the heptanucleotide context explains >81% of vari­
ability in substitution probabilities and was used to
derive substitution intolerance scores for genes and a
new intolerance score for amino acids72. We have devel-
oped a similar approach to the nucleotide structure of
the non-coding genome to define patterns of constraint
and essentiality. Here, the heptameric structure of the
genome defines a context-dependent tolerance score
(CDTS)73 that identifies the constrained regions in the
human non-coding genome. Different from the scores
described above, CDTS is derived uniquely from human
population data and does not use any a priori knowledge
on classes of variation.
In parallel with human genomic studies, the
non-coding genome is increasingly studied by using
CRISPR–Cas9 screens. Fulco et al.74 assessed >1 Mb of
sequence near two essential transcription factors, MYC
and GATA1, and identified nine distal enhancers and
repressors that control gene expression. Korkmaz et al.75
targeted Cas9 to transcription factor binding sites in 758
enhancer regions. Specifically, the study characterized
the role of functional enhancer elements in mediating
p53 and ERα gene regulation. In total, they identified
six enhancer elements that potentially control cell pro-
liferation. Sanjana et al.76 targeted >700 kb surrounding
the genes NF1, NF2 and CUL3 to identify non-coding
locations that modulate drug resistance in melanoma.
Zhu et al.77 reported a CRISPR–Cas9 screen of 700
long non-coding RNAs (lncRNAs). They identified
51 lncRNAs that can positively or negatively regulate
human cancer cell growth. At present, the non-coding
genome CRISPR–Cas9 screens are regional and, based
on their experimental readouts, may not be considered
actual screens of essentiality. In the future, genome-wide
screens will generate data on hallmarks of essentiality
in the non-coding genome. Much as we have reviewed
the essentiality of different regions of the protein-coding
genome, it will be useful to compare data from CRISPR–
Cas9 in vitro screens with human population data of
essentiality in the non-protein-coding genome.
Conclusions
This article highlights approaches for the identification
of genes that can be considered essential in humans,
and emphasizes the importance of rare loss‑of‑­function
variants leading to haploinsufficiency. Evaluating gene
essentiality by leveraging population genomics and
empirical model systems goes beyond the study of gene
function and redundancy. For clinical medicine, these
essentiality metrics serve to support improved attri­
bution of pathogenicity to variants. For drug develop­
ment, the metrics provide an in vivo glimpse on the
impact of natural, lifelong loss‑of‑function variants
when present in a heterozygous or homozygous state78.
One study 54 indicated that drugs that target genes
that tolerate rare homozygous loss‑of‑function have a
greater registration approval rate compared to all other
targets. This would suggest that it is easier or safer to
target mutation-tolerant genes than essential, intoler-
ant genes. For example, development of C‑C chemokine
receptor type 5 (CCR5) antagonists, for the inhibi-
tion of human immunodeficiency virus (HIV) entry
into cells, was supported by the lack of adverse con-
sequences of a natural truncation (CCR5Δ32) variant
that is observed in up to 3% of the human population79.
A second scenario is exemplified by the identification
of heterozygous truncating variants of the gene encod-
ing cytotoxic T lymphocyte antigen 4 (CTLA4). Loss
of this inhibitory receptor on immune cells leads to
severe immune dysfunction through dysregulation
of FOXP3+ regulatory T cells and hyperactivation of
effector T cells80. CTLA4 is one of the current targets
for immune-checkpoint-blocking antibodies in cancer.
The observation of disease in long-term CTLA4 defi-
ciency in natura describes the possible spectrum of drug
toxicity that can be anticipated from CTLA4-blocking
drugs80. A third scenario illustrates aspects of balancing
the expectation from natural impacts of null variants,
with the outcome of clinical trials that antagonize the
target gene. A recent study of evolocumab, a mono­
clonal antibody that inhibits proprotein convertase
subtilisin–kexin type 9 (PCSK9) and lowers low-density
lipoprotein cholesterol levels, reported a 20% reduction
in cardiovascular death over 48 weeks of treatment 81. In
comparison, lifelong carriage of PCSK9 loss‑of‑function
alleles is associated with as much as 50% reduced risk of
myocardial infarction.

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Haplotype phasing
The assignment of an allele to
one of the two copies of the
chromosomes (maternal and
paternal).
We have also emphasized differences between defi-
nitions of essentiality of human genes in cell lines or
knockout mice compared to evidence gathered from
human population genetic studies. Whereas various
sets of essential genes may inform biology and guide
drug development efforts — in particular for targeting
tumour cell viability — the reader should be aware of the
considerable differences in what is scored as essential
between in vitro and in vivo screens, in humans and in
mice (TABLE 2). One approach to mitigating these funda-
mental differences of defining essentiality is to increase
attention to the phenotypic traits of loss‑of‑function
heterozygous mice and, more generally, to measure
the biological consequences of haploinsufficiency. This
may also be important for drug development, as haplo­
insufficiency may be equated to the half maximal inhib-
itory concentration (IC50), a measure of the effectiveness
of a drug in early preclinical development.
Progressively, the metrics and scores used to define
essential genes should serve to narrow down and
identify the actual essential exons and codons in a
gene82. Analyses may also include haplotype phasing,
to support the identification of compound hetero­
zygosity at essential genes. It is important to note that
because deleterious alleles act synergistically, purify-
ing selection limits linkage disequilibrium between
rare loss‑of‑­function variants in the genome83. In the
future, it will be informative to extend the analysis of
gene essentiality to cancer in vivo, as opposed to analyses
in tumour cell lines. For this purpose, large databases
of tumour somatic variation can be studied using the
scores that have been developed for germline defini-
tion of essentiality. Finally, as the understanding of the
hallmarks of essentiality extends to the non-coding
genome, the field will be informed about the possible
consequences of disruption of the essential regulatory
machinery of genes and pathways. Collectively, human73
and interspecies conservation and constraint 84, func-
tional characterization67 and genetic evidence85 build a
map of human genome essentiality.

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Acknowledgements
The authors thank Drs Ewen Kirkness and Michael Hicks for
valuable comments. The authors are employees of Human
Longevity, Inc.
Author contributions
All authors substantially contributed to discussion of content
and to reviewing/editing the manuscript before submission.
I.B., J.d.I. and A.T. researched data for the article and contrib‑
uted to writing the manuscript.
Competing interests statement
The authors declare competing interests: see Web version for
details.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
FURTHER INFORMATION
Cohort Study of Lausanne (CoLaus): http://www.colaus.ch/
Database of Essential Genes (DEG):
http://www.essentialgene.org
dbSNP: https://www.ncbi.nlm.nih.gov/projects/SNP/
Exome Aggregation Consortium (ExAC):
http://exac.broadinstitute.org
Exome Sequencing Project (ESP):
http://evs.gs.washington.edu/EVS/
Fathmm variant effect predictor:
http://fathmm.biocompute.org.uk/
Loss‑Of‑Function Transcript Effect Estimator (LOFTEE):
https://github.com/konradjk/loftee
Mouse Genome Informatics (MGI):
http://www.informatics.jax.org
SnpEff variant effect predictor:
http://snpeff.sourceforge.net/
UK10K Project: https://www.uk10k.org/
VEP-LOFTEE variant effect predictor:
http://uswest.ensembl.org/info/docs/tools/vep/index.html
SUPPLEMENTARY INFORMATION
See online article: S1 (box) | S2 (table) | S3 (table)
ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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