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a
Nobel laureate.
b
For CRISPR milestones, see Table 3.
transplantation of a DNA fragment from a bacteriophage into the SV40 genome [19]. Next, an
Escherichia coli plasmid was used as a vector for inserting a DNA fragment containing a
penicillin resistance gene from another bacterium (Staphylococcus aureus); upon transformation
(A) Type II-Restriction nuclease (B) Type IIS-Restriction nuclease (C) Homing endonuclease
Natural
DA RT
Trends in Biotechnology
Figure 2. Endonucleases as gene and genome editing tools. Top row: Natural endonucleases. (A) Type II restriction
enzyme (e.g., homodimeric EcoRI, 6 bp). (B) Type IIS restriction enzyme (e.g., homodimeric FolkI, 5 bp). (C) Homing endo-
nuclease (e.g., homodimeric CreI, 2x9 bp). Middle row: Synthetic FokI-based nucleases. (D) Zinc finger nuclease (2 × 9
bp). (E) TALEN (2 × 12 bp). (F) CRISPR-Cascade-FokI (2 × 30 bp). dCas9-FokI has also been developed (not shown). Bottom
row: Natural and synthetic CRISPR-Cas9 variants. (G) Cas9 (20 bp). crRNA and tracrRNA are linked with synthetic loop (dotted
circle; sgRNA). An analogous Cas12a (20 bp; only crRNA, no tracrRNA) tool has also been developed (see text). (H) nCas9-
base editor, by fusion of effector to deaminase (DA) variants, such as cytidine deaminase [C-to-U(T)] and adenosine deaminase
[A-to-I(G)]. dCas12a base editors have also been developed (not shown). Yellow arrowhead indicates base edit of nucleotide
(s) on nontarget strand. (I) nCas9-Prime Editor, by fusion of effector to reverse transcriptase and using an extended sgRNA
(see text for details). General: red arrowheads indicate strand cleavage.
to E. coli cells, the gene was functionally expressed, allowing the recombinant E. coli strain to
grow on penicillin-containing agar plates [20]. In a follow-up experiment, the same approach
was used to express genes from a frog in E. coli [21] (Table 1).
Around the same time, major technical advances were made, including gel chromatography
and DNA sequencing [23,24]. By combining the new insights in biology/biochemistry with the
spectacular technological progress (Table 1), the stage was set for a new phase to take off: the
development of molecular biotechnology with unprecedented applications!
Molecular biotechnology
After the aforementioned pioneering fundamental studies on recombination of homologous and
heterologous DNA fragments, many academic and industrial research groups realized the potential
of genome editing tools for fundamental and applied goals. One of the most spectacular examples
of those days was the functional expression of mammalian somatostatin (a peptide hormone of
14 amino acids [25]) in E. coli. A next challenge was the production of recombinant human insulin,
a hormone consisting of two peptides: the A chain (21 amino acids) and the B chain (30 amino
acids) [26]. The two peptide-encoding genes were chemically synthesized, and each was fused to
the E. coli lacZ gene that was integrated in an expression plasmid. Next, the two recombinant
plasmids were transformed in parallel to two E. coli hosts. After cultivation, the fusion proteins were
purified, the insulin peptides were cleaved off their LacZ carrier, and the two mature chains were
combined and covalently bound through cysteine bridges, resulting in the first human drug created
by recombinant DNA technologies [26]. These first examples of functional polypeptide products
generated by chemically synthesized genes represent a major biotechnological breakthrough.
A few years later, another milestone in the molecular biology field was the groundbreaking discov-
ery of the polymerase chain reaction (PCR) by Kary Mullis (1983) [27] (Table 1). The impact of PCR
on the development of both fundamental and applied research has been truly overwhelming. The
PCR method enabled the exponential amplification of any DNA fragment [27], allowing efficient
cloning procedures, including genome and transcriptome analyses (see following text). In addi-
tion, PCR boosted the development of specific and sensitive diagnostics, such as for disease-
related mutations (e.g., sickle cell anemia [27]) and disease-causing pathogens (e.g., severe
acute respiratory syndrome coronavirus 2 [28]). Moreover, many variations on the PCR theme
have been developed over the years, such as allowing the introduction of site-directed mutations
at any site of an amplicon and for generating fusions of two or more PCR products (from synthetic
genes to synthetic genomes, see following text).
Apart from rational mutagenesis, also random PCR-based approaches have been developed over
the years. Whereas classical mutagenesis is based on physical, chemical, or biological methods
that randomly generate genetic variation of biological systems in vivo, error-prone PCR allows
in vitro mutagenesis of DNA fragments, either by using low-fidelity DNA polymerases or by performing
the amplification reaction under suboptimal conditions. This PCR application has been instrumental
for generation of genetic libraries that allow laboratory evolution [29,30] (Table 1), in which the princi-
ples of natural evolution (genetic variation, expression, and selection) have been used in a laboratory
setting, resulting in an acceleration of the process with spectacular results (reviewed by [30,31]).
As mentioned previously, the first examples of genome editing concern the in vitro plasmid
assembly (cut and paste) and subsequent transformation to E. coli, as well as the in vivo
site-directed mutagenesis of an E. coli ssDNA phage [22]. Likewise, seminal analyses of the
interaction between E. coli and the dsDNA phage lambda revealed three different recombina-
tion mechanisms by which the chromosome of the bacteriophage could integrate into the
chromosome of its bacterial host (reviewed by [41]): the E. coli RecABCD system for homolo-
gous recombination (HR), the lambda-encoded Int-Xis pathway for site-specific integration,
and the lambda Red system for HR. In the following section, the three recombination mechanisms
are described in more detail, with a focus on features that are relevant for genome editing
applications.
The bacterial RecA protein plays a central role in DNA repair through HR, just as its orthologs in
archaea (RadA) and eukaryotes (Rad51). In case of a DNA double-strand break (DSB), the
RecBCD complex initially processes the flanks of the cut. The RecBCD exonuclease activity
results in partial generation of ssDNA, after which RecA monomers associate to form long
ssDNA-bound RecA filaments that scan dsDNA for regions homologous to the ssDNA. Upon
recognition of a matching sequence, RecA catalyzes homology-directed strand invasion of the
dsDNA helix, eventually resulting in repair of the DSB [42]. The activity of RecA-mediated HR
may vary substantially, depending on the organism, the growth conditions, and/or the phase of
the cell cycle. In case of host cells with a relatively active HR system (e.g., S. cerevisiae), plasmids
or linear DNA fragments can be used for delivery of to-be-integrated DNA. The location of integra-
tion depends on the flanking regions of the used DNA constructs. For host cells with a relatively
low HR activity, genomic inserts often include a selection marker [e.g., antibiotic resistance (AbR)]
that, after selection, can be eliminated from the recombinant cell, e.g., using the Cre-lox system
(see following text) (Table 2). Another way to optimize the screening of recombinants is the use of
a
Abbreviations: ABE, adenine base editors; CBE, cytosine base editors; MAGE, multiplex automated genome engineering; SNV, single-nucleotide variant.
b
Cargo sizes: small <0.1 kb; medium 0.1–1 kb, large >1 kb. Flanking sequences vary 0.05–1.0 kb. Marker, generally antibiotic resistance. Counterselection: original
sequence is selected against by CRISPR-Cas-based targeting (toxic DSB) or by releasing cI-based inhibition of antibiotic resistance.
c
CRISPR-editing independent of double-strand break (DSB) formation.
d
An integrated marked flanked with Cre-motifs can be removed (marker-free) with Cre recombinase.
e
Insertion of a serine recombinase motif (e.g., attB) may be an option for other recombination strategies and/or when integration of large cargo is desired.
counterselection, such as by CRISPR-Cas (discussed later). In cases in which the latter system
does not result in recombinants (e.g., in nonmodel organisms with very low HR efficiency), the
recently developed SIBR-Cas technology can ‘buy time’ for the desired recombination to occur
before inducing counterselection with CRISPR-Cas. SIBR-Cas is based on the disruption of
the gene encoding a Cas nuclease by synthetic variants of a self-splicing intron, the activity of
which can be induced, resulting in well-controlled timing of counterselection [43] (Table 2).
The Integrase (Int) enzyme is a key player of the site-specific recombination pathway of phage
lambda. Integrase is a serine recombinase that catalyzes the specific crossover between the
21–25-bp motifs on the phage genome (attP) and on the bacterial genome (attB), resulting in
site-specific integration of phage lambda in the E. coli chromosome. This system has been
used to develop the Gateway cloning system for efficient transfer of a DNA fragment between
different expression vectors, in which the Int enzyme and the excisionase (Xis) enzyme are
used for integration and excision, respectively [44] (Table 2).
Cre-lox is a similar system derived from E. coli phage P1, also used for recombination in eukaryotes.
Unlike the counterpart from phage lambda, the Cre recombinase catalyzes both the site-specific
integration and excision [45].
The lambda-Red system (Red-αβγ) has been demonstrated to be a very useful tool for HR in
E. coli. The required lambda-Red proteins are generally encoded by an expression plasmid in
the bacterial host [46,47]. This ‘recombineering’ tool allows the integration of homologous
ssDNA or dsDNA to obtain a desired genome edit (insertion, substitution, or deletion) in several
bacteria and even in some eukaryotes (reviewed by [41,48]). In case ssDNA oligonucleotides
are used as a mutagen, they are designed to base pair (as ‘synthetic’ Okazaki fragments) with
the complementary lagging strand of the chromosomal replication fork. After introduction of the
ssDNA into the cell, ssDNA is bound to lambda Red-β, an ssDNA-annealing protein (SSAP).
This SSAP interacts specifically with a host ssDNA-binding protein (SSB) that is associated
with the lagging strand of the replication fork. This interaction results in delivery and annealing
of the ssDNA oligo to the lagging DNA strand (Table 2). An interesting variation on this
recombineering theme is MAGE, which allows multiplex engineering by introducing multiple oligo-
nucleotides simultaneously [49,50]. For using the recombineering system in organisms other than
E. coli, screening efforts have resulted in several RecT variants that perform better, most likely due
to a better match with the hosts’ SSB protein [49] (Table 2).
has recently been reported in which counterselection is based on the initial genomic integration of
a DNA fragment that encodes the robust lambda cI inhibitor, which blocks the expression of an
adjacent toxin gene, as well as the expression of an antibiotic resistance (AbR) marker on the
lambda-Red plasmid. Only upon replacement of the cI-toxin cassette by a fragment of interest
(supplied separately) is the repression of the AbR gene on the lambda-Red plasmid relieved,
hence allowing strong selection for the desired integration. This lambda-Red recombineering
system has been reported to allow integration of markerless, operon-sized cassettes with effi-
ciencies between 50% and 100% [52] (Table 2).
The first system that was demonstrated to use a nucleic acid guide to target a complementary nucleic
acid was the Argonaute protein. As part of the eukaryotic RNAi pathway, the eukaryotic Argonaute
(eAgo) variants use short RNA guides to find a matching mRNA sequence, resulting either in transcript
binding (silencing of expression) or in transcript cleavage. This system is currently used as an RNAi
tool to treat selected genetic diseases in human patients [59]. Interestingly, when studying prokaryotic
Argonaute (pAgo) proteins, variants were found that use DNA or RNA guides to target DNA, raising
the possibility for using them as novel genome editing tools [60,61]. A potential advantage of DNA
targeting pAgos is that they do not use a PAM-like sequence (see following text, CRISPR-Cas), but
a disadvantage of these pAgos is that they do not use a PAM; this drawback relates to the relatively
poor ability of Argonaute proteins to target dsDNA [62]. However, several recent studies have
reported the potential of pAgo variants for genome editing, at least in bacteria [63–65]. In addition,
thermostable pAgos have potential for diagnostics of disease-related sequences [66].
Last but not least, a second group of proteins that use nucleic acid guides to target complemen-
tary nucleic acids are members of the CRISPR-Cas family (clustered regularly interspaced short
palindromic repeats and associated protein). Since their discovery [67–69], the family tree of
CRISPR-Cas systems has grown substantially. Natural CRISPR-Cas variants that have been
detected in (meta)genomes are divided into two classes, each with three types and dozens
of subtypes [70]: CRISPR class 1 with large multiprotein cascade(-like) complexes (type I/
cascade-Cas3, type III/Csm/Cmr and type IV) and CRISPR class 2 with smaller multidomain
proteins (type II/Cas9, type V/Cas12, and type VI/Cas13). The only common feature
between all Cas effectors is the use of CRISPR-derived RNA (crRNA) guides. Cas effectors
use different ways to bind their guide, either by using a hairpin/pseudoknot structure at one
of the crRNA ends as an anchor (e.g., Cascade, Cas12a) or by binding a second tracrRNA that
partly base pairs with a flanking sequence of the crRNA (e.g., Cas9, Cas12b). In addition, apart
from the major differences in overall architecture between class 1 and class 2 effectors, major dif-
ferences in size occur in class 2 nucleases, roughly ranging from 400 to 1400 amino acids, which
most likely reflects the evolutionary stages from a transposon ancestor to nucleases with increased
structural complexity [71]. Another major difference is the target of the different systems: dsDNA in
case of Cascade/Cas3 [72,73], Cas9 [74], and Cas12 [75] and RNA in case of Cmr/Csm [76] and
Cas13 [77]. Recently, some Cas12 variants have been described that are catalytically inactive,
either as a subunit of a guided transposition system (Cas12k [78]) (Table 2) or as systems that
act by silencing the expression of target genes (Cas12c and Cas12m [71,79]). Since the demon-
stration that the system functions as a bacterial adaptive immune system [80], key features of the
molecular mechanism were elucidated at a rapid pace (Table 3). Table 3 shows major discoveries
in CRISPR research, from the discovery of the repetitive arrays to sophisticated synthetic variations
for precision engineering. As a basis for both the molecular understanding of the DNA-targeting
CRISPR systems as well as for their genome editing applications, essential discoveries concern
the crRNA guide processing and architecture, allowing the straightforward design of crRNA guides
for adjusting the Cas effectors to attack complementary DNA targets [72,75,81], the protospacer
adjacent motif (PAM) sequence and position (up/down-stream the protospacer sequence)
[75,82,83], and the DNA cleavage site location [73–75]. Although the need for a PAM motif does
restrict the number of potential guides, the advantage of the PAM recognition mechanism is that
it triggers efficient guide invasion of the dsDNA helix; as mentioned before, this contrasts with the
PAM-independent Argonaute nucleases.
In conclusion, specific DNA DSBs can be generated in chromosomes of prokaryotes and eukaryotes
by using different DNA-targeting enzymes: meganucleases (Figure 2A), FokI-derived nucleases
(Figure 2B), Argonaute nucleases, or CRISPR-associated nucleases (Figure 2C). Importantly, the
outcome of editing at the site of the introduced break depends on the type of repair system that is
responsible for restoring the integrity of the chromosome. Depending on the cell to be edited (and
its growth phase), repair can be executed by homologous repair systems and/or by nonhomologous
repair systems. For homologous-directed repair (HDR), a repair template (ssDNA or dsDNA) is
generally used to introduce desired precision edits to close the break; the HDR system is often
expressed only during replication of genomic DNA, i.e., in growing cells. The nonhomologous end-
joining systems usually generate small insertions and/or deletions around the break, whereas the
microhomology-mediated end joining mainly generates deletions through recombination of short
homologous regions surrounding the break. The latter systems are used mainly for gene inactivation.
Details on the impact of repair systems on genome editing have recently been reviewed [84].
As Cas9 was the first available CRISPR tool (Figure 2G), many attempts have been made to fur-
ther optimize its performance. Engineering efforts have focused on obtaining Cas9 variants with
enhanced editing precision and efficiency, either by using rational residue substitutions or by lab-
oratory evolution. More concretely, this has resulted mainly in optimization of the guide (sgRNA),
the PAM requirements, and the specificity (reviewed by [94,95]). In addition, in order to avoid un-
desired genetic recombination upon the introduction of chromosomal DSBs, DSB-independent
strategies have been developed in which synthetic fusions of Cas9 nickases (nCas9) are used,
initially with cytidine and adenosine deaminases (base editors) [94,96] (Figure 2H). A next
milestone was the development of prime editors (PEs), which are fusions of nCas9 with a reverse
transcriptase (RT) equipped with an extended prime editing guide (pegRNA) (reviewed by [95])
(Figure 2I). A recent variation on this theme is the twinPE method, in which an nCas9-PE protein
and two pegRNAs are designed to bind on opposing strands of a selected genomic locus. After
nicking and primed extension, this may result in substitution of the DNA sequence between the
PE-induced nick sites. When combined with a serine (or tyrosine) recombinase (e.g., Int, Cre;
described above), twinPE can be used to introduce the 34–50-bp recombinase attachment
motif (e.g., attB, lox), allowing targeted integration of DNA fragments of thousands of base
pairs [97] (Table 2). A similar approach includes programmable addition via site-specific targeting
elements, which uses an nCas9-PE fused to a serine integrase (Bxb1), which was capable of
inserting up to 36-kb DNA cargos into specific genomic loci of human cell lines [98].
Another CRISPR-based application is the use of catalytic inactive Cas proteins (e.g., dead Cas9;
dCas9 and derivatives) for binding a target promoter or open reading frame. Such binding inhibits
transcription initiation or transcription elongation by RNAP, resulting in the silencing of the target
gene due to the reduced production of mRNA transcripts [99,100]. Another form of CRIPSR-
based gene silencing is targeting mRNA transcripts for cleavage by using Cas13 variants [77],
a process that resembles that of Argonautes (see above) and commonly referred to as CRISPR
interference (CRISPRi). An alternative to Cas13-mediated silencing is Cas13-mediated base
editing by using a catalytically inactive Cas13 (dCas13) fused to an adenosine deaminase acting
on RNA (ADAR) base editor [101–103]. This approach can be used for correcting point mutations
on the transcriptome level without interfering with the host genome. In a similar fashion, dCas
proteins fused to RNAP-recruiting factors (e.g., ω subunit of RNAP) have been used to activate
the expression of a gene by binding to sequences adjacent to promoters and by favoring the
recruitment of RNAP at the target locus [99,100]. Activation of a target gene by CRISPR-Cas
approaches is therefore termed ‘CRIPSR activation’ (CRISPRa). CRISPRi and CRISPRa,
although they are not genome editing tools per se (in that they alter the genome of the host),
they are important tools for gene characterization and metabolic engineering.
An important requirement for therapeutic purposes is the appropriate delivery of the editing tools. For
in vivo gene therapy, the adeno-associated virus (AAV) is considered a promising delivery strategy.
However, because the big cas9 gene exceeds the limited cargo capacity of AAV, a ‘combinatorial-
deletion’ approach has recently been used to minimize Cas9 from 1368 to 874 amino acids [104].
Cas12 variants may, at least in some cases, be attractive alternatives for Cas9. Favorable features
of Cas12a include their autonomous guide processing, implying a short crRNA guide (no tracrRNA)
and multiplex editing using design guides derived from a compact CRISPR array. Cas12 nucleases
typically generate DSBs that are positioned distant from its PAM, a feature that may result in
enhanced levels of HR [75]. Although Cas12a was found to be very specific, its editing efficiency
has been reported to be relatively low. A laboratory evolution approach recently resulted in a highly
efficient ‘Cas12a Ultra’ variant for editing in human cells while maintaining its high on-target specificity
[105]. Another interesting feature concerns the size of several Cas12 subtypes, especially the
recently discovered minimal natural Cas12 variants [70,106], because this may allow AAV delivery.
Moreover, attempts to use the aforementioned Cas12k-associated transposons for site-directed
insertion of large DNA fragments in hosts other than E. coli have so far been unsuccessful. However,
a recently discovered missing link concerns a (dual-function) protein from E. coli, which may allow its
use in other (eukaryotic) organisms as well [107].
Last, apart from the aforementioned Argonaute-based diagnostics application [66], a range of
CRISPR-based diagnostic tools have been developed in recent years, allowing accurate detec-
tion of (disease-related) single-nucleotide variants as well as monitoring of bacterial and viral
pathogens. The CRISPR sensors are based on different effectors: Cas9 (‘LEOPART’, [108]),
Cas12 (‘DETECTR’, [109]), Cas13 (‘SHERLOCK’, [110]), and Cmr [111] (‘SCOPE’ [112]).
Altogether, the availability of an intriguing collection of nucleases with easily programmable RNA
guides and purpose-specific functionalities provides an excellent basis for a new generation of
genome editing tools.
impact of CRISPR is certainly reflected by scientific articles describing basic research; as of Outstanding questions
December 2022, a PubMed search with the term ‘CRISPR’ resulted in >35 000 hits, steadily Can we develop genome editing tools
increasing in the period 2002–2022. that are accurate and efficient, and
that can be delivered at specific
tissues, to allow for in vivo gene
In terms of CRISPR-related products that reached the market, microbial biotechnology (‘white therapy?
biotech’) is the forerunner. This is based mainly on the production and sale of Cas proteins that
are used for fundamental and applied research in industry and academia. Although CRISPR- How will academia, companies and
governments resolve the complex IP
Cas technologies are widely used for engineering of microbial cell factories, the current patent
situation with genome editing tools, es-
situation is probably a major hurdle for its commercialization. In plant biotechnology (‘green bio- pecially the IP related to CRISPR-Cas?
tech’), a recent breakthrough was the release of a CRISPR-edited tomato with elevated levels
of GABA (claimed to lower blood pressure) in September 2022 on the Japanese market [114]. What will regulatory bodies do to
speed up the commercialization of
Another spectacular study showed the power of CRISPR editing for the domestication of the CRISPR-generated products, and
wild tomato [115]. In addition, many examples of CRISPR-edited crops (rice, wheat, and how will the regulatory bodies distin-
maize) in which relevant features have been improved are reported in the literature. However, guish and regulate CRISPR-based ge-
to the best of our knowledge, these crops are not (yet) on the market [116–119]. nome editing to previous ‘classical
mutagenesis’?
As for medical applications (‘red biotech’), it is important to highlight the use of CRISPR-Cas to How will scientists, regulators and
create mutant cell lines that are used to decipher the role of genetic factors in disease and devel- public figures communicate to the
general public the potential, societal
opment. As to gene therapy, the ongoing CRISPR-based Cas9/Cas12a-related therapeutic
and commercial benefits of genome
approaches are still in early-stage clinical trials, with approval anxiously awaited. Most of these editing?
trials concern ex vivo editing of hematopoietic stem cells (blood and immune cell precursors) either
to treat hemoglobin-related genetic diseases (e.g., sickle cell disease, β-thalassemia) or to engineer
defense cells to establish an immune therapy to treat different types of cancer [120–123]. A recent
milestone (December 2022) for CRISPR-based therapeutics was achieved when a patient with
T-cell leukemia was cured of cancer by base editing of donated T cells ex vivo [124]. The donated
T cells underwent a series of modifications, including disabling the T cell targeting mechanism to
avoid attacking the recipient’s body, removal of the CD7 marker to protect the engineered T
cells from the recipient’s immune system, and genomic alterations to resist chemotherapy. This
world-first use of CRISPR-Cas-mediated base editing therapy has been called a potential
‘scientific layup’ for the approval of other promising therapies [125]. In addition, clinical trials have
been initiated in which, for the first time, in vivo editing is executed in human patients with a hered-
itary eye disease [126]. Moreover, as mentioned above, several Argonaute and CRISPR-based
tools have been repurposed as tunable sensors both for disease-related mutations and for
monitoring of pathogenic bacteria and viruses [66,108–112]. On the basis of spectacular progress
in molecular understanding and technological developments, we are currently in the middle of a
genome editing revolution. The impact of CRISPR-Cas cannot be overstated: It not only has
major commercial potential, but, above all, it has great societal promise. Together with revolutionary
progress in adjacent research areas (including technologies for synthesis and sequence analysis of
DNA, as well as prediction algorithms for gene function and protein structure [127–129]), CRISPR-
based technologies are anticipated to contribute substantially to improve sustainable production,
pathogen detection, curing of certain heritable genetic diseases, and food security. However,
before the full potential of CRISPR-Cas can be exploited, there are still some hurdles to overcome:
technical, commercial, and societal.
Although big steps have been made to improve the performance of CRISPR tools (e.g., precision
and efficiency), a major technical issue relates to the delivery of editing systems for in vivo thera-
peutic applications (see Outstanding questions). Next, the commercialization of the technology
needs a boost. A major factor that may slow down applications appears to be the complex intel-
lectual property (IP) landscape and (at least in the European Union) the regulation of CRISPR
editing of crops. To realize the aforementioned promises, it is crucial that the IP situation will be
clarified and that regulation will be adjusted on the basis of a proper risk assessment. Last
but not least, societal acceptance of the new editing technologies is of utmost importance,
especially in case of food-related applications. Only if there is general consensus that genome
editing is in the best interest of society as a whole can it be further developed. If that is the case,
we can prepare for a future as foreseen by the Committee of the Nobel Prize Chemistry 1993
(https://www.nobelprize.org/prizes/chemistry/1993/press-release/) when describing the impact
of the molecular biology discoveries of the laureates Kary Mullis and Michael Smith: ‘The future
also holds possibilities of gene therapy, curing hereditary diseases by specifically correcting
mutated code words in the genetic material. Site-directed mutagenesis of plant proteins is opening
up the possibility of producing crops that can make more efficient use of atmospheric carbon
dioxide during photosynthesis.’ We conclude that this possibility is definitely getting closer, and it
is expected that at least some of the dreams of 30 years ago may become a reality soon!
Acknowledgments
The authors acknowledge the Dutch Research Council (NWO Spinoza grant SPI 93-537 and NWO Gravitation grant
024.003.019), and the European Research Council (ERC-AdG-834279) for financial support.
Declaration of interests
J.v.d.O. is cofounder of NTrans Technologies. J.v.d.O. is a scientific advisory member of the Scientific Advisory Board of
NTrans Technologies, Scope Biosciences, and Hudson River Biotechnology. Wageningen University has patents issued
and/or pending for CRISPR technologies on which J.v.d.O. and C.P. are inventors.
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