RESEARCH

RESEARCH

HPV Primary Cervical Screening: Making the Switch from Cytology to HPV testing

Cervical screening has employed the Papanicolaou (Pap) test for over 80 years to detect cervical pre-cancer and cancer in women. The Pap test can be considered one of the most effective disease prevention strategies, preventing countless cases and mortalities associated with cervical cancer. However, it has its limitations, even with modern improvements such as liquid based cytology, sampling, sensitivity and screening subjectivity has reported up to a 40% false negative rate globally. The Human Papillomavirus (HPV) has long been known to be the primary aetiological factor in
the pathogenesis of cervical cancer and there are 14 known high-risk HPV subtypes [16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68]. Of these HPV 16 and 18 are associated with ~70% of all cervical cancer cases.

HPV testing has been used with great success in Ireland to triage low-grade cytology screening results since 2015. It has also been used as a test of cure following treatment and in the management of uncertainty at colposcopy since 2012 and 2014. More recently, using HPV rather than Cytology as the primary screening test has been shown in numerous international randomised control trials to have a significantly higher sensitivity and negative predictive value and based on this evidence, organised screening programmes worldwide are making the switch to HPV primary screening.

Following the HIQA “Heath technology assessment of HPV testing as the primary screening method for cervical screening” in 2017, The Irish Cervical Screening Programme, CervicalCheck, had committed to making this switch. In preparation for HPV primary screening, Coombe Women and Infants University Hospital, which houses the only remaining accredited Public Hospital Cervical Cytology Screening Laboratory in the country, had begun a programme of readiness for HPV testing and recently received ISO 15189 accreditation for HPV DNA testing on the Roche Cobas 4800 platform.

The core concept of HPV primary screening is to detect all women with high-risk HPV and thus those women at risk of developing cervical pre-cancer or cancer. Essentially, HPV primary screening is a test of risk for cervical malignancy, rather than of disease. But what about the high prevalence of HPV in our cervical screening population and are all HPV positive women at equal risk? The HPV primary screening approach will require that a positive HPV result is further triaged to take that test of risk into a test of disease. While many triage options exist e.g. p16/Ki67 staining of cytology smears and host gene methylation markers, with some of them being more informative than others, it has been recommended that the Pap test which has served us well so far is used to further stratify HPV positive women. There is no doubt that the addition of further risk stratification tests or the expansion of cytology as a “test of disease” will present opportunities to enhance the specificity of HPV primary screening and further refine the definition of risk.

In the future, we may see a more tailored approach, where the triage test for a HPV positive woman at the beginning of her cervical screening journey is different to a woman near exiting the programme, with HPV vaccination status also a factor. After nearly 80 years since the introduction of the Pap test cervical screening is set to dramatically change for the better with innovation and good science leading the way forward to a better cervical screening programme.

Dr Stephen Reynolds, Dr Helen Keegan, Dr Christine White, Roisin O’Brien,
Stephen Dempsey, Dr Cara Martin, Martina Ring, Prof. John O’Leary.
– on Behalf of the Cytopathology Dept. Coombe Women and Infants University Hospital, Dublin

Scientific Background on the Nobel Prize in Chemistry 2020

TOOL FOR GENOME EDITING

The Royal Swedish Academy of Sciences has decided to award Emmanuelle Charpentier and Jennifer A. Doudna the Nobel Prize in Chemistry 2020, for the development of a method for genome editing.

Introduction
In 1953, J.D. Watson and F.H.C. Crick reported the molecular
structure of DNA [1]. Ever since, scientists have tried to develop
technologies that can manipulate the genetic material of cells and
organisms. With the discovery of the RNA-guided CRISPR-Cas9
system, an easy and effective method for genome engineering
has now become a reality. The development of this technology
has enabled scientists to modify DNA sequences in a wide range
of cells and organisms. Genomic manipulations are no longer an
experimental bottleneck. Today, CRISPR-Cas9 technology is used
widely in basic science, biotechnology and in the development of
future therapeutics [2].
The discovery of the CRISPR-Cas system in
prokaryotes.
The work that eventually led to the discovery of the powerful
CRISPR-Cas9 system for genome editing began with the
identification of repeated genome structures present in bacteria
and Archaea. In 1987, a report noted an unusual repeated
structure in the Escherichia coli genome, which contained five
highly homologous sequences of 29 base pairs (bp), including
a dyad symmetry of 14 bp that were interspersed by variable
spacer sequences of 32 bp [3]. Some years later, similar, repeated
structures were identified in the genome of the halophilic Archaea
Haloferax mediterranei, with 14 almost perfectly conserved
sequences of 30 bp, repeated at regular distances [4].
Subsequent bioinformatics analyses revealed that these types of
repeats were common in prokaryotes and all contained the same
peculiar features: a short, partially palindromic element occurring
in clusters and separated by unique intervening sequences of
constant length, suggesting an ancestral origin and high biological
relevance [5]. The term CRISPR was introduced, an abbreviation for
clustered regularly interspaced short palindromic repeats [6].
An important step towards understanding the function of CRISPR
came with the identification of CRISPR-associated (cas) genes,
a group of genes only present in CRISPR-containing prokaryotes
and always located adjacent to CRISPRs. The identified cas genes
encoded proteins with helicase and nuclease motifs suggesting a
role in DNA metabolism or gene expression [6]. The association
with CRISPR was used as a defining characteristic and over the
coming years a number of Cas protein subfamilies were described
[7, 8].
The functional importance of the CRISPR loci remained elusive
until 2005, when researchers noted that the unique CRISPR
sequences were derived from transmissible genetic elements,
such as bacteriophages and plasmids [9-11]. Prokaryotes carrying
these specific sequences appeared protected from infection, since
plasmids or viruses containing a sequence matching a spacer
(named protospacers) were usually absent in the prokaryote
carrying the spacer [9, 11].
These correlative findings suggested a function for CRISPRs
in prokaryotic defence against invading foreign DNA and the
spacer sequences were described as a ‘memory of past “genetic

aggressions”’ [10]. It had already been shown that CRISPRs were
transcribed into long RNA molecules (pre-crRNA), which were
subsequently processed by cleavage within the repeat sequences
to yield small CRISPR-RNAs (crRNAs) [4, 12]. Taken together these
observations indicated that crRNA could play a role in targeting viral
nucleic acids, perhaps in a manner similar to RNAi in eukaryotic
cells. It was also hypothesized that the Cas proteins was involved in
this process [9].
Later research has indeed demonstrated that crRNA binds to
one or more Cas proteins to form an effector complex that targets
invading nucleic acids. Extensive efforts during the past 25 years
have identified a number of different CRISPR-Cas systems, which
are now divided into two major classes [13]. In the Class 1 systems,
specialised Cas proteins assemble into a large CRISPR-associated
complex for antiviral defence (Cascade). The Class 2 systems are
simpler and contain a single multidomain crRNA-binding protein
(e.g. Cas9) that contains all the activities necessary for interference.
CRISPR-Cas functions as an adaptable
defence system
The hypothesis that CRISPR-Cas systems could confer resistance
to invading foreign DNA was verified in 2007 [14]. In an elegant
set of experiments, scientists studied a Class 2 system in a strain
of Streptococcus thermophilus, which they infected with virulent
bacteriophages. Next, bacteria resistant to infection were isolated
and their CRISPR loci analysed. The experiment revealed that
resistant bacteria had acquired new spacer sequences, which
matched sequences within the infecting phage used to select
resistance. Deletion of the spacer region led to loss of resistance,
and the phages that were able to grow on resistant bacteria
had accumulated mutations in the protospacer sequence in the
phage genome. Furthermore, inactivation of one of the cas genes
(cas5) resulted in loss of phage resistance. The experiments
thus demonstrated a role for cas gene products in CRISPR-Cas–
mediated immunity and that the specificity of the system was
dependent on the spacer sequences [14].
Further insights into the function of CRISPR-Cas came from
investigations of E. coli, which contains a Class 1 CRISPR-Cas
system encoding no less than eight different Cas proteins. Five of
these gene products could be purified as a multiprotein complex
termed Cascade (CRISPR-associated complex for antiviral defence).
Cascade was shown to function in pre-crRNA processing, cleaving
the long transcripts in the repeated regions and thereby producing
shorter crRNA molecules containing the virus-derived sequence
[15]. After cleavage, the mature crRNA molecules were retained
by Cascade, and, assisted by a cas-encoded helicase, Cas3, they
served as guide molecules that enabled Cascade to interfere with
phage proliferation. The results thus suggested two different steps
in CRISPR function: first, CRISPR expression and crRNA maturation,
and second, an interference step that required the Cas3 protein. The
results also provided evidence suggesting that the E. coli CRISPRCas
system targets phage DNA and not RNA, inasmuch as crRNA
with complementarity to either of the two DNA strands could interfere
with phage proliferation [15].
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Conclusive evidence for DNA being the target of CRISPR-Cas
interference came from elegant experiments using a strain of
Staphylococci epidermidis that contained a CRISPR array with a
spacer sequence homologous to a gene present in a conjugative
plasmid [16]. Transfer of the plasmid into the strain occurred only if
the spacer sequence was mutated or deleted. A self-splicing intron
was inserted into the target sequence on the plasmid. In this way,
the CRISPR spacer would be complementary not to the DNA, as it
is disrupted by an intron, but to the RNA, which would be spliced,
reconstituting the sensitive target. Indeed, insertion of the selfsplicing
intron was sufficient to overcome CRISPR-Cas inhibition
of plasmid transfer, strongly implicating DNA as the primary target
[16]. This conclusion was further supported from studies of S.
thermophilus, in which the CRISPR-Cas system was shown to
cleave both bacteriophage and plasmid DNA in vivo [17].
Protospacer adjacent motifs distinguish
CRISPR from invading DNA.
If spacers lead to cleavage of DNA with matching sequences,
how do they avoid cleaving their own CRISPR spacers? The
answer to this question came from studies of sequences around
protospacers, i.e. the sequences in the phage genomes that had
given rise to spacers. Short sequence motifs were noted just a
couple of nucleotides away from protospacer sequences [11, 18].
These motifs were later labelled protospacer adjacent motifs or
PAMs [19].
The functional importance of PAMs became clear from work
studying the phage response to CRISPR-encoded resistance in S.
thermophilus. In these studies, phages that had overcome bacterial
resistance were isolated and analysed. These studies revealed
that a number of those resistant to CRISPR immunity had acquired
mutations in the PAMs, implicating these short sequences as
important for targeting [20]. Later studies have demonstrated that
the PAM sequences are required both for target interference and for
uptake of new spacer sequences into CRISPRs [21, 22].
Discovery of the CRISPR-Cas9 system
By 2011, it was clear that CRISPR-Cas systems were widespread
in prokaryotes and functioned as adaptive immune systems to
combat invading bacteriophages and plasmids (Figure 1). Studies
had also established that the Cas proteins functioned at three
different levels: (i) integration of new spacer DNA sequences into
CRISPR loci, (ii) biogenesis of crRNAs, and (3) silencing of the
invading nucleic acid [23, 24].
The identification of CRISPR-Cas9 as a tool for genomic editing
came from studies of the Class-2, Type-II CRISPR-Cas system in
S. thermophilus and the related human pathogen Streptococcus
pyogenes. This system contains four cas genes, three of which
(cas1, cas2, csn2) are involved in spacer acquisition, whereas
the fourth, cas9 (formerly named cas5 and csn1), is needed for
interference [14]. In support of this notion, inactivation of the cas9
gene prevented cleavage of target DNA [17]. To further define the
elements required for immunity, the S. thermophilus CRISPR-Cas
system was introduced into E. coli, where it provided heterologous
protection against infection with phages and plasmids [25]. Using
this experimental model, parts of the system were inactivated to
define the components required for protection. The work clearly
demonstrated that the Cas9 protein alone was sufficient for the
CRISPR-encoded interference step, and that two nuclease domains
present in the protein, HNH and RuvC, were both required for this
effect [25].
Discovery of tracrRNA and its role in crRNA
maturation
In 2011, Emmanuelle Charpentier and colleagues reported

Figure 1. A general scheme for the function of the CRISPR-Cas
adaptive immune system as presented in [26]. Three stages
are identified. Adaptation: Short fragments of double-stranded
DNA from a virus or plasmid are incorporated into the CRISPR
array on host DNA. crRNA Maturation: Pre-crRNA are produced
by transcription and then further processed into smaller
crRNAs, each containing a single spacer and a partial repeat.
Interference: Cleavage is initiated when crRNA recognize and
specifically base-pair with a region on incoming plasmid or virus
DNA. Interference can be separated both mechanistically and
temporally from CRISPR acquisition and expression.

on the mechanisms of crRNA maturation in S. pyogenes [27].
Using differential RNA sequencing to characterize small, noncoding
RNA molecules, they identified an active CRISPR locus,
based on expression of pre-crRNA and mature crRNA molecules.
Unexpectedly, the sequencing efforts also identified an abundant
RNA species transcribed from a region 210 bp upstream of the
CRISPR locus, on the opposite strand of the CRISPR array (Figure
2a).
The transcript was denoted trans-encoded small RNA (tracrRNA)
and contained a stretch of 25 nucleotides (nt) with almost perfect
complementarity (1-nt mismatch) to the repeat regions of the
CRISPR locus, thus predicting base pairing with pre-crRNA [27].
The RNA duplex region that would form included processing sites
for both pre-crRNA and tracrRNA, which immediately suggested
that the two RNAs could be co-processed upon pairing (Figure 2b).
In support of the proposed idea, deletion of the tracrRNA locus
prevented pre-crRNA processing and vice versa. Charpentier
and colleagues also noted that a co-processed duplex involving
tracrRNA and pre-crRNA would have short 3′ overhangs, similar to
those produced by the endoribonuclease RNase III, and they went
on to demonstrate that this enzyme could process a heteroduplex
formed between tracrRNA and pre-crRNA in vitro and was required
for tracrRNA and pre-crRNA processing in vivo. Finally, the
researchers found that processing also involved the Cas9 protein,
since deletion of the cas9 gene in bacteria impaired both tracrRNA
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Figure 2. Identification of tracrRNA in S. pyogenes as reported in [27]. a. Differential RNA sequencing (dRNA-seq) reveals
expression of tracrRNA and crRNAs. Sequence reads of cDNA libraries of RNA are shown on top. Below is the genomic organisation
of tracrRNA and CRISPR01/Cas loci. Red bar: tracrRNA is encoded on the minus strand and detected as 171-, 89- and ~75-nt
tracrRNA species. Black rectangle inside the red bar: 36-nt sequence stretch complementary to CRISPR01 repeat. The pre-crRNA is
encoded on the plus strand. Black rectangles: CRISPR01 repeats; green diamonds: CRISPR01 spacers; 511, 66 and 39-42 nt: precrRNA
and processed crRNAs. b. Base-pairing of tracrRNA with a CRISPR01 repeat is represented. Cleavages observed by dRNAseq
and leading to the formation of short overhangs at the 3′ ends of the processed RNAs are indicated by two black triangles.

and pre-crRNA processing. Based on their findings, Charpentier
and coworkers suggested that the Cas9 protein acts as a molecular
anchor that facilitates base pairing between tracrRNA and precrRNA,
which in turn allows recognition and cleavage by the host
RNase III protein [27].
Previous reports had revealed the importance of Cas9 for
interference. Charpentier and Jennifer A. Doudna initiated a
collaboration to investigate if crRNA could be used to direct the
sequence specificity of the nuclease. In contrast to what had been
hypothesised in Charpentier’s report a year earlier, addition of
crRNA to purified Cas9 could not stimulate Cas9-catalysed target
DNA cleavage [27, 28].
At this point, the two scientists made a crucial discovery. Addition
of tracrRNA to the in vitro reaction triggered Cas9 to cleave the
target DNA molecule. The tracrRNA thus had two critical functions:
triggering pre-crRNA processing by the enzyme RNase III and
subsequently activating crRNA-guided DNA cleavage by Cas9.
In a series of in vitro biochemistry experiments, the researchers
investigated the biochemical mechanisms of the reaction [28].
The two nuclease domains in Cas9, HNH and RuvC, were each
shown to cleave one strand of target DNA. Cleavage occurred 3
bp upstream of the PAM sequence, which in S. pyogenes has the
sequence 5′-NGG-3′, with N corresponding to any of the four DNA
bases. Furthermore, as predicted from previous reports, target
recognition and cleavage were inhibited by mutations in the PAM
sequence [20].
A peculiar aspect of PAM sequence dependence was that
cleavage of double-stranded DNA was sensitive to mutations in
both the complementary and non-complementary strand whereas
cleavage of single-stranded DNA targets was unaffected by
mutations in the PAM motif. These observations led the authors
to conclude that PAM motifs may be required to allow duplex
unwinding [28].
Similar findings were also published in another report using the
related CRISPR-Cas system in Streptococcus thermophilus. As in
Charpentier and Doudna’s work, this report also demonstrated
that Cas9 cleaves within the protospacer, that cleavage specificity
is directed by the crRNA sequence, and that the two nuclease
domains within Cas9, each cleave one strand. However, the
researchers did not notice the crucial importance of tracrRNA for
sequence-specific cleavage of target DNA [29].
In their study, Charpentier, Doudna and colleagues also worked

to delineate the regions of tracrRNA and crRNA that are absolutely
required for Cas9-catalysed cleavage of target DNA. This led to the
identification of an activating domain in tracrRNA and the realisation
that a “seed region” of ~10 nt in the PAM-proximal region of the
target strand was especially important for target recognition.
Based on their in vitro biochemical analysis, the authors
hypothesized that the structural features in the two RNA molecules
required for Cas9-catalysed DNA cleavage could be captured in a
single RNA molecule. In a crucial experiment, they demonstrated
that this was indeed possible: the RNA components (crRNA and
tracrRNA) of the Cas9 complex could be fused together to form an
active, chimeric single-guide RNA molecule (sgRNA).
Furthermore, Charpentier and Doudna demonstrated that
the sequence of the chimeric sgRNA could be changed so that
CRISPR-Cas9 would target DNA sequences of interest, with the
only constraint being the presence of a PAM sequence adjacent to
the targeted DNA. They had thus created a simple two-component
endonuclease, containing sgRNA and Cas9, that could be
programmed to cleave DNA sequences at will.
The importance of this finding was not lost on them. In the
abstract of the paper reporting their findings, the authors wrote:
“Our study reveals a family of endonucleases that use dual-RNAs
for site-specific DNA cleavage and highlights the potential to exploit
the system for RNA programmable genome editing” [28].
A molecular understanding of the CRISPR
mechanism
Today, there is a detailed structural understanding of how the Cas9-
gRNA complex recognizes its target and mediates cleavage. This
information has been important for efforts to engineer new versions
of the system, with altered PAM specificity and reduced off-target
activities [30].
The structure of Cas9 in free form revealed two distinct lobes,
the recognition (REC) lobe and the nuclease (NUC) lobe, with the
latter containing the HNH and RuvC nuclease domains. When Cas9
binds to sgRNA, it undergoes a structural rearrangement, with the
REC lobe moving towards the HNH domain (Figure 3).
For target recognition, the 20-nt spacer sequence must form
complementary base pairs with the protospacer sequence. In the
structure of Cas9 in complex with sgRNA, the 10-nt seed sequence
in the spacer adopts an A-form conformation and is positioned
Page 3

to engage with the target sequence in DNA [31, 32]. The seed
sequence is located in the 3′ end of the 20-nt spacer sequence and
is essential for target recognition [25, 28, 33]. In genome editing,
similarities between the seed sequence and genome sequences
can cause off-target effects, even if there are many mismatches
elsewhere in the spacer region of sgRNA [34].
As noted, a PAM sequence must also be present next to the
target site, and mutations in this motif prevent Cas9-dependent
cleavage at the target sequence. The Cas9 protein first searches
for the PAM sequence, and once found, probes the flanking DNA
for complementarity to the sgRNA. The GG dinucleotides in PAM
are recognized by base-specific hydrogen-bonding interactions
with two arginine residues in a PAM interacting site, which is
disordered in the apo-form of Cas9, but becomes ordered after
sgRNA binding. The interactions between PAM and Cas9/sgRNA
lead to destabilization of the adjacent double-stranded DNA, which
in turn facilitates for sgRNA to invade the double-stranded DNA.
The destabilization is in part explained by a kink in the target DNA
strand, which is caused by Cas9 interactions with the phosphate
group immediately upstream of the PAM in the same strand [22].
Once a stable RNA–DNA duplex, an R-loop, has been formed,
Cas9 is activated for DNA cleavage. Each of the two nuclease
domains cleaves one strand of the target double-stranded DNA
at a specific site 3 bp from the 5′-NGG-3′ PAM sequence, and in
most cases, the ends that are formed are blunt. By inactivating one
of the two domains, a nickase can be formed, i.e. an enzyme that
cleaves only one strand of a DNA duplex [28, 29]. Nickases are
very useful for practical applications of CRISPR-Cas systems, since
they can be programmed to target opposite strands and thus make
staggered cuts within the target DNA. In this way, a Cas9 nickase
mutant, combined with a pair of sgRNA molecules, can introduce
targeted double-strand breaks with very high sequence specificity
[35].
The application of the CRISPR-Cas9
technology in higher cells
Genome editing relies on the existence of natural pathways for DNA
repair and recombination. Double-stranded breaks typically lead
to either non-homologous end joining (NHEJ) repair or homologydirected
repair (HDR). In the case of NHEJ, the ends are directly
ligated back together and the process usually results in a small

insertion or deletion of DNA at the break, frequently causing frame
shifts in coding sequences and loss of protein expression. The HDR
pathway instead uses a homologous DNA sequence as a template
to repair the break. By introducing modified genetic sequences
as templates for the HDR, it is thus possible to introduce defined
genomic changes such as base substitutions or insertions.
DNA can be introduced into mice embryonic stem cells and
recombine there with the matching sequence within the host
genome to produce gene-modified animals. This method is
powerful but labour-intensive, since recombination events are rare
and require a selectable marker, such as an antibiotic resistance
gene, to be identified. Recombination efficiency is enhanced if
a double-stranded break is introduced at the site of the desired
recombination event, which led to a search for endonucleases that
can be programmed to cleave DNA at locations of interest.
An important earlier step in the engineering of sequencespecific
nucleases came with the development of zinc finger
nucleases (ZFNs) and transcription activator–like effector nucleases
(TALENs). When linked to a nuclease domain, zinc finger proteins
can function as site-specific nucleases that can cleave genomic
DNA in a sequence-specific manner and stimulate site-specific
recombination [36, 37]. TALENs provide yet another DNA-binding
modality that recognizes DNA in a modular fashion and that can
be fused to nuclease domain [38]. Both ZFNs and TALENs are
powerful tools for genome editing. However, their widespread
use has been limited by the inherent difficulties of protein design,
synthesis and validation.
In their work, Charpentier and Doudna defined a simple
two-component system that could rapidly be programmed for
sequence-specific cleavage of target DNA and thereby sparked a
revolution in genome editing. The first experimental demonstration
that CRISPR-Cas9 could indeed be harnessed for genome editing
in human and mouse cells came in early 2013 [39, 40]. These
influential studies demonstrated that Cas9 nucleases could be
directed by crRNA of a defined sequence to induce precise
cleavage at endogenous genomic loci in mouse and human cells.
For the reaction to occur, tracrRNA, crRNA, and Cas9 were all
required, whereas RNase III was replaced by endogenous enzyme
activities.
Just as observed by Charpentier and Doudna in vitro, the
system could be further simplified in vivo, and a chimeric sgRNA
molecule together with Cas9 was sufficient to cleave target

Figure 3. A schematic representation of the mechanism by which CRISPR-Cas9 recognizes
and targets DNA for cleavage as presented in [30]. Binding of sgRNA leads to a large conformational
change in Cas9. In this activated conformation, the PAM-interacting cleft (dotted circle), becomes prestructured for PAM sampling, and the seed sequence of sgRNA is positioned to interrogate adjacent DNA for complementarity to sgRNA.
The process starts with PAM recognition, which in the next step leads to local DNA melting and RNA strand invasion. There is a step-wise elongation of the R-loop formation and a conformational change in the HNH domain to ensure concerted DNA cleavage.
Abbreviations: bp, base pair; NUC, nuclease lobe; PAM, protospacer adjacent motif; REC, recognition
lobe; sgRNA, single-guide RNA.

 

 

Page 4

Figure 4. Genome editing with Cas9 as presented in [48]. a. The Cas9
enzyme is directed to target DNA by a guide RNA and produces a
double-stranded break. A piece of DNA can be used as a template for
homology-directed repair (HDR). b. Cas9 can be fused to a deaminase
enzyme. The mutant Cas9 produces a nick, which stimulates deaminase
activity. The deaminase converts a cytidine base (C) to uracil (U).
DNA repair then repairs the nick and converts a guanine–uracil
(G–U) intermediate to an adenine–thymine (A–T) base pair. c. Prime
editing. A nick-producing Cas9 and a reverse transcriptase enzyme
produce nicked DNA, into which sequences corresponding to the
guide RNA have been incorporated. The original DNA sequence is cut
off, and DNA repair then fixes the nicked strand to produce a fully
edited duplex.

DNA. The system has also been used to introduce genome modification in a number of other eukaryotic systems [41], including Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, Danio rerio and Arabidopsis thaliana [42-46], demonstrating its broad applicability.
In ongoing work, scientists are trying to expand the usefulness of the CRISPR-Cas system for genome editing. In addition to Cas9 from S. pyogenes, a number of other Cas homologues are used today for genome editing and related purposes. Naturally occurring CRISPR systems have other PAM requirements, and in addition, new Cas9 variants are continually engineered to have altered PAM compatibilities. CRISPR-Cas systems can also be used to target RNA. Studies of Pyrococcus furiosus demonstrated that in this species, the system encodes for a crRNA-guided Cas complex, which targets foreign mRNA [47]. Efforts are also under way to develop evermore precise CRISPRCas–based genome editing strategies [48]. These efforts include strategies for base editing at specific sites in eukaryotic genomes (Figure 4). As an example, a cytidine deaminase enzyme has been
fused to a mutant form of Cas9 that cleaves only one strand – a
nickase. When programmed with sgRNA for the desired sequence,
this system can be targeted to a specific genomic location, induce
a nick in the DNA there, and mediate the direct conversion of
cytidine to uridine, which after replication results in a cytosine-tothymine
conversion [49].
Another elegant example is a method called prime editing, in
which a Cas9 nickase is fused to a reverse transcriptase enzyme
[50]. In this approach, the sgRNA contains an additional piece of
RNA, creating a “prime editing guide RNA” that both specifies the
target site and encodes the desired edit. Once produced by the
reverse transcriptase, the DNA synthesized can be installed at the
nick, replacing one of the original DNA sequences.
Concluding remarks
In 2012, Charpentier and Doudna reported “that the Cas9
endonuclease can be programmed with guide RNA engineered as
a single transcript to cleave any double-stranded DNA sequence”
[28]. Their discovery has led to widespread applications of the
CRISPR-Cas9 system as a powerful and versatile tool in genome
editing.

By introducing a vector encoding the Cas9 nuclease and an engineered sgRNA, scientists are now able to make precise singlebase-pair changes or larger insertions. Coupled with the availability of genome sequences for a growing number of organisms, the technology allows researchers to explore these genomes to find out what genes do, move mutations that are identified as associated with disease into systems where they can be studied and tested for treatment, or where they can be tested in combinations with other mutations. The technology has enabled efficient targeted
modification of crops and is currently being developed to treat and cure genetic diseases, for instance by modifying hematopoietic stem cells to treat sickle cell disease and ß-thalassemia.
Finally, it should be emphasised that the power of the CRISPRCas9 technology also raises serious ethical and societal issues. It is of utmost importance that the technology is carefully regulated and used in responsible manner. To this end, the World Health Organization has recently established a global multi-disciplinary expert panel to examine the scientific, ethical, social and legal challenges associated with human genome editing, with the aim to develop a global governance framework for human genome editing.
Claes Gustafsson
Professor of Medical Chemistry
Member of the Royal Swedish Academy of Sciences
Member of the Nobel Committee for Chemistry
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