ArticlePDF AvailableLiterature Review

CRISPR-Cas systems: ushering in the new genome editing era


Abstract and Figures

In recent years there has been great progress with the implementation and utilization of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) systems in the world of genetic engineering. Many forms of CRISPR-Cas9 have been developed as genome editing tools and techniques and, most recently, several non-genome editing CRISPR-Cas systems have emerged. Most of the CRISPR-Cas systems have been classified as either Class I or Class II and are further divided among several subtypes within each class. Research teams and companies are currently in dispute over patents for these CRISPR-Cas systems as numerous powerful applications are concurrently under development. This mini review summarizes the appearance of CRISPR-Cas systems with a focus on the predominant CRISPR-Cas9 system as well as the classifications and subtypes for CRISPR-Cas. Non-genome editing uses of CRISPR-Cas are also highlighted and a brief overview of the commercialization of CRISPR is provided.
Content may be subject to copyright.
Full Terms & Conditions of access and use can be found at
ISSN: 2165-5979 (Print) 2165-5987 (Online) Journal homepage:
CRISPR-Cas systems: ushering in the new genome
editing era
Fernando Perez Rojo, Rikard Karl Martin Nyman, Alexander Arthur Theodore
Johnson, Maria Pazos Navarro, Megan Helen Ryan, William Erskine &
Parwinder Kaur
To cite this article: Fernando Perez Rojo, Rikard Karl Martin Nyman, Alexander Arthur Theodore
Johnson, Maria Pazos Navarro, Megan Helen Ryan, William Erskine & Parwinder Kaur (2018)
CRISPR-Cas systems: ushering in the new genome editing era, Bioengineered, 9:1, 214-221, DOI:
To link to this article:
© 2018 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Published online: 03 Jul 2018.
Submit your article to this journal
Article views: 149
View Crossmark data
CRISPR-Cas systems: ushering in the new genome editing era
Fernando Perez Rojo
, Rikard Karl Martin Nyman
, Alexander Arthur Theodore Johnson
Maria Pazos Navarro
, Megan Helen Ryan
, William Erskine
, and Parwinder Kaur
Centre for Plant Genetics and Breeding, School of Agriculture and Environment, The University of Western Australia, Crawley, WA, Australia;
School of BioSciences, The University of Melbourne, Victoria, Australia;
Institute of Agriculture, The University of Western Australia, Crawley,
WA, Australia;
School of Agriculture and Environment, The University of Western Australia, Crawley, WA, Australia;
Telethon Kids Institute,
Subiaco, WA, Australia
In recent years there has been great progress with the implementation and utilization of Clustered
Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) systems
in the world of genetic engineering. Many forms of CRISPR-Cas9 have been developed as genome
editing tools and techniques and, most recently, several non-genome editing CRISPR-Cas systems
have emerged. Most of the CRISPR-Cas systems have been classified as either Class I or Class II and
are further divided among several subtypes within each class. Research teams and companies are
currently in dispute over patents for these CRISPR-Cas systems as numerous powerful applications
are concurrently under development. This mini review summarizes the appearance of CRISPR-Cas
systems with a focus on the predominant CRISPR-Cas9 system as well as the classifications and
subtypes for CRISPR-Cas. Non-genome editing uses of CRISPR-Cas are also highlighted and a brief
overview of the commercialization of CRISPR is provided.
Received 5 March 2018
Accepted 25 April 2018
CRISPR products; Cas9;
Cas13; genome editing;
CRISPR-Cas figure; Cas
classes; Cas types
The CRISPR-Cas system (clustered regularly inter-
spaced short palindromic repeats) has become one
of the most powerful tools in the arsenal of molecular
biologists and geneticists since its discovery by Ishino
et al. in 1987 [1]. Mojica et al. [2]performedmuchof
the initial characterization of CRISPR-Cas systems
during the 1990s and the term CRISPR was coined
for the first time by Jansen et al. in 2002 [3]. Since
then, the discoveries and characterisations of the pro-
teins and molecules involved, as well as the processes
that generally occur across all types of the CRISPR-
Cas system [4]. Using the predominant Class 2, Type
II CRISPR-Cas9 [46]systemasanexample,
CRISPR-Cas systems effectively consist of a three-
stage process: expression, interference and adaptation
[4,5]. During expression, the CRISPR array which
contains many sequences homologous to specific tar-
get sequences (protospacers) are transcribed into
what is called pre-CRISPR RNA (pre-crRNA) [46]
(Figure 1(a)), and these pre-crRNA form homologous
bonds with smaller transactivating crRNAs
(tracrRNA) [46]. Once this complex has formed, it
attaches to a Cas9 protein where the long pre-crRNAs
are cut and separated by RNase III into individual
crRNA/tracrRNA complexes (Figure 1(b)).
Interference begins as the crRNA/tracrRNA guides
the Cas9 complex to a target sequence and the
crRNA binds to the target sequence after the so called
protospacer adjacent motif (PAM) (Figure 1(c)). It is
thisshortsequencethatallowsfor self/nonself-discri-
mination as the sequence is absent from the hosts own
CRISPR array [46].Thetargetsequenceisunwound
at this stage and cut by the Cas9 proteinsnuclease
domains (RuvC and HNH) [46], leaving a double
stranded break in the target DNA sequence, after
which the Cas9 complex detaches. The desired DNA
repair template is then inserted and attached to the
end of the interference by Homology Directed Repair
(HDR). The repaired spacer sequence is then tran-
scribed and adapted into the genome (Figure 1(d))
[46]. Adaptation in the majority of the known
CRISPR-Cas systems is controlled by the Cas1 and
Cas2 proteins (and to some extent Cas4) that adapt
the desired spacer sequences into the CRISPR array by
CONTACT Parwinder Kaur Centre for Plant Genetics and Breeding, School of Agriculture and Environment, The
University of Western Australia, Crawley, WA 6009, Australia.
2018, VOL. 9, NO. 1, 214221
© 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (, which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
integrating the RNA and then inducing reverse tran-
scription of the RNA into DNA [46]. It is by these
processes that some bacteria are able integrate viral
genomes into their own (i.e. the CRISPR array) dur-
ing an infection, thus allowing a more effective
immune response during future infections [47].
More recently these processes have been modified to
function as powerful tools for molecular biology and
genetic engineering. The CRIPSR-Cas system classifi-
cations and developments are reviewed below, along
with of non-genome editingCRIPSR-Cassystemsand
the current state of CRISPR-Cas commercialization.
CRISPR-Cas subtypes and classifications
The ever-evolving interaction between prokaryotes
and the viruses that infect them has resulted in wide
variation among the CRISPR-Cas systems [7]. The
general classification divides the known Cas systems
into two classes, six types, and 19 subtypes. Currently,
they are classified according to the structure shown in
Table 1 [48]. This system is widely distributed in
archaeal (87%) and bacterial (50%) genomes, Class I
being the most commonly found (90%) [57].
The main difference between the classes are how
the effector modules are composed. In Class I, the
effector is comprised of a complex formed by sev-
eral proteins with different functions, whereas in
Class II, the effector is associated with a single
multi-domain protein [4,7]. Considering the inte-
gration module (adaptation step), the proteins that
are involved in this process are Cas1 and Cas2,
which integrate a viral protospacer into the bacter-
ial/archaea genome, and its function remains
Figure 1. a) The crRNA from the CRISPR array combines with a smaller tracrRNA molecule, becoming a gRNA complex. b) The gRNA
binds with a Cas9 protein, forming a gRNA:Cas9 complex. c) The gRNA guides the Cas9 protein, targeting a specific DNA sequence,
which it first recognizes by the PAM motif. The RuvC and HNC nuclease sites cuts the target sequence, leaving two homologous
blunt ends. d) The desired DNA repair template inserts the desired gene and repairs the strands by HDR, the product DNA then
undergoes adaptation into the organisms genome.
conserved throughout classes, apart from Type IV,
in which the processes remain unknown [4,5]. By
contrast, the effector modules involved in the target
recognition and cleavage steps, are generally vari-
able throughout types. The CRISPR-Cas system
classifications are further discussed below. Class I
is divided into three types (Type I, III and IV)
where the effector module is generated by a cascade
of different Cas and other accessory proteins [47].
Class II systems are used more in research and will
be reviewed in further detail below.
Class II CRISPR-Cas classifications
Most researchers have used Class II types to date
because they enable them to work with only one
multidomain protein [4,7]. Within this Class, the
predominantly researched group is Type II, in
which we find the well-known Cas9 effector.
Cas9 is a protein with two nuclease domains
(RuvC and HNH) [6] that requires two combined
RNA molecules to produce a double stranded
break (DSB) with blunt ends in the target DNA
sequence. These RNA molecules are a crRNA, and
a tracrRNA, that together guide the interference.
Researchers have bioengineered these RNA mole-
cules into one guide RNA molecule (gRNA) which
alone contains both functions of the crRNA and
tracrRNA, thereby making CRISPR systems even
easier to utilize [9]. The Cas9 effector spCas9 from
Streptococcus pyogenes is the most widely used
effector due to its high efficiency at producing
DSB. However, spCas9 has three major limitations.
Firstly, the PAM is NGG, which dictates the need
for sequences with two consecutive GG to produce
the DBS, making its use problematic in AT rich
sequences [10]. Secondly, the size of this protein is
1,368 amino acids, which can be a hindrance when
introducing these sequences into viral vectors [11].
Thirdly, Cas9 is prone to producing off target
effects, which means that DSB may be generated
at incorrect locations [12].
Several solutions to these problems have been
tested. One possible solution is to modify the spCas9
sequence to obtain better variants with less off target
effects. This has been progressed through generation
of a highly specific Cas9 that contains mutations that
reduce the interactions between the nuclease domain
and the non-specific DNA: spCas9 HF (high fidelity)
[13]. To overcome the PAM restrictions, the same
spCas9 was engineered to obtain different PAM
motifs to make this tool even more adaptable to dif-
ferent types of DNA sequences, such as VQR, EQR
and VRER [14]. Another alteration to spCas9 is the
removal of one of the nuclease domains. As a result,
nCas9 (nickase Cas9) was generated, nCas9 can
induce a single stranded break, and using two of
these enzymes with two gRNA; a deletion or other
alterations with a reduced amount of off target effects
can be achieved [15]. To solve the problem related to
spCas9s large size, Cas9 homologues from other
organisms can be used. One example, saCas9
(Staphylococcus aureus Cas9) is smaller and has a
different PAM site [16]. Cas9 proteins are continually
being altered to obtain even more variations in size,
site recognition and target effect.
The second type of Class II is the Type V. This
group has the characteristic of sharing the RuvC
Table 1. Overview of CRISPR-Cas classification and subtype defining characteristics [48].
Class I Class II
Type I Type III Type IV Type II Type V Type VI
Cas1/2 [3] Cas1/2 Unknown Cas1/2 Cas1/2 Cas1/2
47 Cas protein Cascade Cas9 [10] Cas12a (cpf1)/Cas12b/
Cas13c [20]
Organism bacteria and archaea archaea bacteria bacteria and archaea bacteria
fused to
HD fused to
unknown RuvC and HNH RuvC and Nuc HEPN domains (2)
tracrRNA no No no yes cpf1-no no
Cleavage motif subtype
dependant (7)
dependant (2)
dependant (2)
CG rich NGG (blunt
AT rich (staggered ends) non-G PFS (ssRNA)
Histidine-aspartate domain
nuclease domain with Type II, but not the HNH
domain. This type is divided in three subtypes (A, B
and C) of which the most cited and used is subtype
A, which includes the Cpf1 effector nuclease (also
referred interchangeable as Cas12a) [6]. This type of
nuclease has four distinctive characteristics that has
allowed it to develop as a complementary product
for the Cas9 effector. Firstly, this type of nuclease
does not need a tracrRNA sequence to be func-
tional, making the design easier and more cost
effective. Secondly, the size of this protein is even
smaller than Cas9, making insertion into viral vec-
tors easier. Thirdly, when this enzyme generates a
cleavage, it leaves staggered ends, improving the
chances of a non-homologous end join (NHEJ)
knock-in. Fourthly, it recognizes AT rich PAM
sites, making this enzyme complementary to Cas9
(CG rich zones). Due to all these features, Cpf1 has
become an extremely useful tool for genome editing
[12,17]. Cpf1 continues to be modified to improve
efficiency and it has been reported to be an excellent
tool for plant editing, even better than Cas9 [18].
The high efficiency of CRISPR-Cas systems and
their relatively ease of use makes it possible to generate
organisms with several mutations in the genome. A
rapid method to obtain a multi-mutated organism is
the use of lentiviral gRNA libraries with several gRNA
that can be integrated into any of the Cas mentioned
above. In this way, an organism can be generated with
multiple mutations in only one generation [18,19].
The last integrant of Class II is the recently discov-
ered Type VI. Its most unusual characteristic is that it
further below, together with other non-genome edit-
ing CRISPR-Cas systems.
Delivery methods for CRISPR-Cas systems
An important consideration is the selection of effective
methods to deliver the CRISPR-Cas system to organ-
isms that are to be mutated. There are several methods
available, and choice largely depends on the character-
istics of the organism to be transfected. In plant mod-
els, plasmids in Agrobacterium tumefaciens are often
used as a vector, whereas in mammalian cells use of
the complex gRNA-Cas9 is preferred [9,18].
Non-genome editing methods of the CRISPR-
Cas systems
RNA-targeting with CRISPR-Cas13 and rCas9
One of the most recent discoveries in CRISPR-Cas is
the Cas13 (Cas13a, Cas13b and Cas13c) Class II, type
VIgroup,describedin2015byShmakovetal.[6]. It
should be noted that Cas13a was previously referred to
as C2c2 (Cas13b = C2c4, Cas13c = C2c7) [6,20,21]and
some literature uses these terms interchangeably.
What separates Cas13 from the other predominant
CRISPR-Cas systems, such as CRISPR-Cas9, is that it
targets single-stranded RNA (ssRNA) rather than
double-stranded (dsDNA), and it tends to cleave
RNA non-specifically (Figure 2(a)) [20,21]. Unlike
most of the previously described systems, Cas13 is
guided by a lone crRNA molecule rather than a
crRNA-tracrRNA complex. Another mechanism
that sets Cas13 apart from Cas-types is its twin
HEPN nuclease domains (Table 1), which generate
blunt ends in the target RNA after cutting [20,21]. As
described by Nakade et al. [12], OConnell et al. and
Nelles et al. [22,23] are working to generate variants of
the CRISPR-Cas9 (CRISPR-rCas9) system that can
target ssRNA similarly to the CRISPR-Cas13 system,
by modifying PAM-presenting oligonucleotides
(PAMmers). These PAMmers will navigate the Cas9
to bind specifically to target ssRNA sequences [22,23].
CRISPRa/CRISPRi, epigenetic modifications and
Lundh et al. [24] demonstrated that the Cas9 pro-
tein can be enzymatically deactivated (dCas9) to
lose its ability to cleave while retaining ability to
target and bind to specific DNA sequences. This
dCas9 protein can then be combined with activa-
tor- or repressor domains to systematically activate
or repress upstream genes, which is a reversible
process as the genome is not directly edited [24
27]. A simple model is presented in Figure 2(b); an
activator or repressor domain attaches to the
dCas9 complex, resulting in the activation (and
thus transcription) or the repression of one or
several upstream genes. This system is called
CRISPRa when an activator domain is used, and
CRISPRi when a repressor domain is used [24
27]. These techniques have been developed into
useful genetic screening tools [28,29]. CRISPR-
dCas9 also has another use: epigenetic modifica-
tion. By attaching the dCas9 complex to known
epigenetic modifiers such as histone demethylase
(LSD1) or human acetyltransferase (p300) dCas9
can target the genome with great proficiency.
What differentiates this mechanism from other
CRISPR-Cas systems, is that the dCas9-LSD1 com-
plex works on the chromatin, while the genome is
still wrapped up in histones. Thus, these modifica-
tions are useful tools for heritable gene expression.
These complexes can also serve to activate or
repress transcription, e.g. LSD1 repress pluripo-
tency maintenance genes (e.g. Oct4 and Tbx3) in
mouse embryonic stem cells, which is visualized
in Figure 2(c) [25].
Both the Cas9 and Cas13 systems have been
modified by researchers to function as genetic
markers; dCas9 for DNA and dCas13 for RNA.
For example, Chen et al. [30] demonstrated that a
dCas9 complex tagged with an enhanced green
fluorescent protein (eGFP) can be guided by a
gRNA to target sequences that will then fluoresce
during dynamic imaging (Figure 2(d)) [30,31].
CRISPR-Cas commercialization status quo
In contrast to other genome editing techniques
such as zinc finger nucleases (ZFNs) [32] and
transcription activator-like effector nucleases
(TALENs) [33], CRISPR was originally developed
inside academic research institutions [34]. In 2012,
Jennifer Doudna and Emmanuelle Charpentier
Figure 2. a) CRISPR-Cas13 targets ssRNA with its crRNA, and the twin HEPN nuclease domains cleaves the sequence non-specifically
after the first crRNA guided cleavage at the binding site, leaving blunt ends. b) The dCas9 combines with an activator/repressor
domain to activate/repress an upstream gene, resulting in transcription of that gene into RNA or blocked transcription. c) dCas9-
LSD1 complex targets the genome at the chromatin to repress transcription of the targeted gene by demethylation. d) CRISPR-
dCas9-EGFP as a fluorescent marker complex for imaging.
from the University of California, Berkeley, pub-
lished a paper and initiated their patent applica-
tion which demonstrated the use of CRISPR-Cas9
system to edit DNA [35]. By the end of that year,
another group led by Feng Zhang at the Broad
Institute of MIT (Massachusetts) and Harvard in
Cambridge, initiated another patent which
demonstrated the application of CRISPRCas9 in
mammalian cells [19,36]. Their paper was pub-
lished in 2013, and it initiated a conflict as to
which group would have the rights to CRISPR-
Cas9 intellectual property. This issue currently
remains unresolved, and four additional research-
ers have now also claimed rights to this system
[37]. Since its development, the number of patents
related to CRISPR products has increased at an
unprecedented rate compared to other editing
technologies; several private commercialisations
have been generated in a short period of time
[34]. In 2015 there was a 5-fold increment in
investment in CRISPR and biotechnology compa-
nies received a total of $1.2 billion in venture
capital funds [34]. The spread of this technology
to the private sector has occurred in two ways.
Firstly, the original developers have generated
their own companies, for example Caribou
Biosciences by Charpentier, CRISPR Therapeutics
by Doudna and Editas by Feng Zhang. Secondly,
numerous leading biotechnology companies have
developed new market opportunities with the
technology, such as AstraZeneca, DuPont,
Novartis, Thermo Fisher Scientific and Sigma
Aldrich and several others, and entered into the
market [38].
FDA regulations on the use of CRISPR products
remain unclear in relation to oncological trials,
and it may take several years to obtain final
approvals [34]. However, the situation in agricul-
tural sciences is clearer and some knock-out and
mutated crops have been approved as non-GMO
products in the USA [39]. While the issue regard-
ing who can claim the CRISPR-Cas9 original
patent remains unresolved, CRISPR is well placed
to be commercialized by companies, and to be
further developed by researchers.
The discovery, characterization and development of
CRISPR-Cas systems constitutes a major milestone
for molecular biology in the 21st century. The cur-
rent state of these systems, and furthermore their
future potential as ever more easy-to-use variants
are developed, promises to open many doors for
genetic engineering both in the areas of genome
editing and non-genome editing. Further research is
necessary to fully map out all the molecular
mechanisms involved in the classes and subtypes
(e.g. Class I, Type IV in Table 1). There also remain
limitations to some of the existing systems, such as
CRISPR-Cas9, but recent discoveries have bypassed
several of these limitations and more are under
development. The CRISPR-Cas patent disputes will
eventually be resolved, which may or may not
change the availability and cost of commercially
available CRISPR-Cas systems. Regardless of how
the patent disputes are resolved, CRISPR-Cas sys-
tems will play major roles in a wide range of areas in
the near future including genetic engineering and
screening, mammalian gene therapy and plant and
livestock breeding.
We acknowledge the resources provided by the Centre for
Plant Genetics and Breeding (PGB) at The University of
Western Australia (UWA) for the Masters studies conducted
by Fernando Perez Rojo and Rikard Karl Martin Nyman.
Author contributions
PK, FPR and RKMN conceived and designed the research.
FPR and RKMN performed the literature search, prepared
the figures and wrote the manuscript with contributions from
AATJ, MPN, MHR, WE and PK. All authors read and
approved this manuscript.
Disclosure statement
The authors declare that they have no competing interests.
Fernando Perez Rojo
Rikard Karl Martin Nyman
Alexander Arthur Theodore Johnson
Maria Pazos Navarro
[1] Ishino Y, Shinagawa H, Makino K, et al. Nucleotide
sequence of the iap gene, responsible for alkaline phospha-
tase isozyme conversion in Escherichia coli, and identifica-
tion of the gene product. J Bacteriol. 1987;169:54295433.
[2] Mojica FJM, Diez-Villaseñor C, García-Martínez J,
et al. Intervening sequences of regularly spaced prokar-
yotic repeats derive from foreign genetic elements. J
Mol Evol. 2005;60:174182.
[3] Jansen R, Embden JDA, Gaastra W, et al. Identification
of genes that are associated with DNA repeats in pro-
karyotes. Mol Microbiol. 2002;43:15651575.
[4] Mohanraju P, Makarova KS, Zetsche B, et al. Diverse
evolutionary roots and mechanistic variations of the
CRISPR-Cas systems. Science. 2016;353:aad5147.
[5] Jackson SA, McKenzie RE, Fagerlund RD, et al.
CRISPR-Cas: adapting to change. Science. 2017;356:
[6] Shmakov S, Smargon A, Scott D, et al. Diversity and
evolution of class 2 CRISPRcas systems. Nat Rev
Microbiol. 2017;15:169182.
[7] Makarova KS, Wolf YI, Alkhnbashi OS, et al. An
updated evolutionary classification of CRISPRcas sys-
tems. Nat Rev Microbiol. 2015;13(11):722.
[8] Hille F, Charpentier E. CRISPR-Cas: biology, mechan-
isms and relevance. Philos Trans R Soc Lond B Biol Sci.
[9] Zhang J-H, Adikaram P, Pandey M, et al. Optimization
of genome editing through CRISPR-Cas9 engineering.
Bioengineered. 2016;7(3):166174.
[10] Deltcheva E, Chylinski K, Sharma CM, et al. CRISPR
RNA maturation by trans-encoded small RNA and
host factor RNase III. Nature. 2011;471(7340):602607.
[11] Fu Y, Foden JA, Khayter C, et al. High-frequency off-
target mutagenesis induced by CRISPR-Cas nucleases
in human cells. Nat Biotechnol. 2013;31(9):822826.
[12] Nakade S, Yamamoto T, Sakuma T. Cas9, Cpf1 and C2c1/
2/3Whats next? Bioengineered. 2017;8(3):265273.
[13] Kleinstiver BP, Pattanayak V, Prew MS, et al. High-fide-
lity CRISPR-Cas9 nucleases with no detectable genome-
wide off-target effects. Nature. 2016;529(7587):490495.
[14] Kleinstiver BP, Prew MS, Tsai SQ, et al. Engineered
CRISPR-Cas9 nucleases with altered PAM specificities.
Nature. 2015;523(7561):481485.
[15] Ran FA, Hsu PD, Lin CY, et al. Double nicking by
RNA-guided CRISPR Cas9 for enhanced genome edit-
ing specificity. Cell. 2013; 155:479480.
[16] Ran FA, Cong L, Yan WX, et al. In vivo genome
editing using staphylococcus aureus Cas9. Nature.
[17] Zetsche B, Gootenberg JS, Abudayyeh OO, et al. Cpf1
is a single RNA-guided endonuclease of a class 2
CRISPR-Cas system. Cell. 2015;163:759771.
[18] Scheben A, Wolter F, Batley J, et al. Towards CRISPR/
Cas crops bringing together genomics and genome
editing. New Phytol. 2017;216(3):682698.
[19] Cong, L, Ran FA, Cox D, et al. Multiplex genome
engineering using CRISPR/Cas systems. Science. 2013;
[20] Abudayyeh OO, Gootenberg JS, Essletzbichler P, et al.
RNA targeting with CRISPRcas13. Nature. 2017;550
[21] Cox DBT, Gootenberg JS, Abudayyeh OO, et al. RNA
editing with CRISPR-Cas13. Science. 2017;358
[22] OConnell MR, Oakes BL, Sternberg SH, et al.
Programmable RNA recognition and cleavage by
CRISPR/Cas9. Nature. 2014;516(7530):263266.
[23] Nelles DA, Fang MY, OConnell MR, et al.
Programmable RNA tracking in live cells with
CRISPR/Cas9. Cell. 2016;165(2):488496.
[24] Lundh M, Pluciñska K, Isidor MS, et al. Bidirectional
manipulation of gene expression in adipocytes using
CRISPRa and siRNA. Mol Metab. 2017;6:13131320.
[25] Lo A, Qi L. Genetic and epigenetic control of gene
expression by CRISPRcas systems. F1000. 2017;6:
Faculty Rev747.
[26] Gilbert LA, Horlbeck MA, Adamson B, et al. Genome-
scale CRISPR-mediated control of gene repression and
activation. Cell. 2014;159:647661.
[27] Qi LS, Larson MH, Gilbert LA, et al. Repurposing
CRISPR as an RNA-guided platform for sequence-speci-
fic control of gene expression. Cell. 2013;152:11731183.
[28] Jost M, Chen Y, Gilbert LA, et al. Combined CRISPRi/
a-based chemical genetic screens reveal that rigosertib
is a microtubule-destabilizing agent. Mol Cel. 2017;68
[29] Kampmann M. CRISPRi and CRISPRa screens in
mammalian cells for precision biology and medicine.
ACS Chem Biol. 2017; 13:406416.
[30] Chen B, Gilbert LA, Cimini BA, et al. Dynamic ima-
ging of genomic loci in living human cells by an opti-
mized CRISPR/Cas system. Cell. 2013;155:14791491.
[31] Ma H, Naseri A, Reyes-Gutierrez P, et al. Multicolor
CRISPR labeling of chromosomal loci in human cells.
Proc Natl Acad Sci USA. 2015;112:30023007.
[32] Klug A. The discovery of zinc fingers and their appli-
cations in gene regulation and genome manipulation.
Annu Rev Biochem. 2010;79:213231.
[33] Li T, Huang S, Jiang WZ, et al. TAL nucleases
(TALNs): hybrid proteins composed of TAL effectors
and FokI DNA-cleavage domain. Nucl Acids Res.
[34] Brinegar K, Yetisen A, Choi S, et al. The commercializa-
tion of genome-editing technologies. Crit Rev Biotechnol.
[35] Jinek M, Chylinski K, Fonfara I, et al. A programmable
dual-RNAguided DNA endonuclease in adaptive bac-
terial immunity. Science. 2012;337(6096):816821.
[36] Ledford H. How the US CRISPR patent probe will play
out. Nat News [Internet]. 2016 Mar 10;531(7593):149.
[cited 2017 Dec 29]. Available from:: http://www.nat
[37] Cohen J. Ding, ding, ding! CRISPR patent fight enters
next round [Internet]. 2017 Jul 26, Am 9: 00. [cited
2017 Dec 29]. Available from: http://www.sciencemag.
REGENHEALTHSOLUTIONS [Internet]. [cited 2017
Dec 29]. Available from: http://www.regenhealthsolu
[39] Wolter F, Puchta H. Knocking out consumer concerns
and regulators rules: efficient use of CRISPR/Cas ribo-
nucleoprotein complexes for genome editing in cereals.
Genome Biol. 2017;18:43.
... It basically includes Cas nuclease、CRISPR-derived RNA and transactivating RNA. Under gRNA guidance, Cas nuclease initiates the targeted cleavage of DNA double strands and participates in gene editing [13]. In comparison with traditional nucleases, RNA-guided CRISPR/Cas9 has the advantages of economic use, convenience, efficiency, and easy operation [14]. ...
Full-text available
Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder involving multiple organ systems. TSC2 gene plays an important role in the development of TSC. The most common kidney manifestation of TSC is renal angiomyolipoma (RAML). TSC-RAML is more likely to be bilateral multiple tumors and tends to destroy the renal structure and damages renal function severely. As a result, patients with TSC-RAML often miss the opportunity for surgical treatment when TSC-RAML is diagnosed, causing difficulty in obtaining tumor specimens through surgery. Due to this difficulty, model cell lines must be constructed for scientific research. In this paper, TSC2 was knocked out in NIH-3T3 cell lines by CRISPR/Cas9 system. PCR, WB and mTOR inhibitor drug sensitivity test showed that the TSC2 knockout NIH-3T3 cells were successfully constructed. The ability of proliferation and invasion in TSC2 KO NIH-3T3 cells were higher than those in wild type group. The constructed KO cell line lay the foundation for further study of TSC.
... Genome editing requires a short protospacer adjacent motif (PAM) sequence near the target, two combined RNA molecules, trans-activating RNA (tracrRNA), and CRISPR RNA (crRNA) to induce double strand breaks (DSBs) with blunt ends in the targeted DNA sequence. These two RNA molecules can be combined into a single guide RNA (gRNA) performing dual functions (Rojo et al., 2018). ...
Inherited retinal diseases (IRDs) are a clinically complex and heterogenous group of visual impairment phenotypes caused by pathogenic variants in at least 277 nuclear and mitochondrial genes, affecting different retinal regions, and depleting the vision of affected individuals. Genes that cause IRDs when mutated are unique by possessing differing genotype-phenotype correlations, varying inheritance patterns, hypomorphic alleles, and modifier genes thus complicating genetic interpretation. Next-generation sequencing has greatly advanced the identification of novel IRD-related genes and pathogenic variants in the last decade. For this review, we performed an in-depth literature search which allowed for compilation of the Global Retinal Inherited Disease (GRID) dataset containing 4798 discrete variants and 17,299 alleles published in 31 papers, showing a wide range of frequencies and complexities among the 194 genes reported in GRID, with 65% of pathogenic variants being unique to a single individual. A better understanding of IRD-related gene distribution, gene complexity, and variant types allow for improved genetic testing and therapies. Current genetic therapeutic methods are also quite diverse and rely on variant identification, and range from whole gene replacement to single nucleotide editing at the DNA or RNA levels. IRDs and their suitable therapies thus require a range of effective disease modelling in human cells, granting insight into disease mechanisms and testing of possible treatments. This review summarizes genetic and therapeutic modalities of IRDs, provides new analyses of IRD-related genes (GRID and complexity scores), and provides information to match genetic-based therapies such as gene-specific and variant-specific therapies to the appropriate individuals.
... org/prizes/chemistry/2020/press-release/) method of genome editing. CRISPR/Cas9 works by retarding the miRNA biogenesis through introduction of INDELs at miRNA processing sites of MIR genes [127,128]. Apart from biogenesis, the INDELs can also hamper miRNA-mRNA target pairing, full deletion and knock-in of the MIR genes and/or their promoters [20]. CRISPR-derived systems, such as dCas9 nickase79, fCas980, Cpf181, Cpf1 and Csm1 are being searched and studied for more flexible, efficient and applicable usage [129].Despite being most used, CRISPR/Cas9 faces some complexities and challenges while editing MIR genes. ...
Full-text available
Global projections on the climate change and the dynamic environmental perturbations indicate severe impacts on food security in general, and crop yield, vigor and the quality of produce in particular. Sessile plants respond to environmental challenges such as salt, drought, temperature, heavy metals at transcriptional and/or post-transcriptional levels through the stress regulated network of pathways including transcription factors, proteins and the small non-coding endogenous RNAs. Amongst these, the miRNAs have gained unprecedented attention in recent years as key regulators for modulating gene expression in plants under stress. Hence, tailoring of miRNAs and their target pathways presents a promising strategy for developing multiple stress tolerant crops. Plant stress tolerance has been successfully achieved through the over expression of microRNAs such as Os-miR408, Hv-miR82 for drought tolerance; OsmiR535A and artificial DST miRNA for salinity tolerance, and OsmiR535 and miR156 for combined drought and salt stress. Examples of miR408 overexpression also showed improved efficiency of irradiation utilization and carbon dioxide fixation in crop plants. Through this review, we present the current understanding about plant miRNAs, their roles in plant growth and stress-responses, the modern toolbox for identification, characterization and validation of miRNAs and their target genes including in silico tools, machine learning and artificial intelligence. Various approaches for up-regulation or knock-out of miRNAs have been discussed. The main emphasis has been given on the exploration of miRNAs for development of bioengineered climate-smart crops that can withstand changing climates and stressful environments, including combination of stresses, with very less or no yield penalties.
... Still, it is undeniable that by harnessing major scientific and technological innovations, the legal and ethical boundaries in that realm have been dramatically pushed (29). In light of the fact that science is moving in uncharted territory as far as such techniques are concerned, it is essential to shed a light on the science that could make embryo editing possible, but also on the legal, ethical, and social ramifications which it entails (30). Such applications are so far banned in virtually all developed countries, but could such restrictions be some day circumvented in the same way bans on surrogacy and other assisted reproductive technologies have been? ...
The paper addresses the issue of the legality and ethical admissi-bility of invasive experiments on embryos and the correlated one of the degree of legal protection and dignity to be recognized for human embryos, particularly in light of the growing importance that scientific research on embryonic stem cells has been gaining from the clinical and biomedical standpoints in the therapeutic treatments of diseases so far considered incurable, in the interest of public health. Furthermore, the issue of experimentation on cryopreserved supernumerary human embryos is still extremely polarizing, which makes it harder to arrive at shared solutions. The author hopes for a broad-ranging debate at the international level, for the ultimate purpose of achieving shared regulatory frameworks.
... Indeed, it would be timely to shift from morpholinos, affecting the whole embryo, to less toxic and more precise cell-specific gene editing tools such as the CRISPR-cas systems, as discussed by e.g. (Rojo et al., 2018;Schulte-Merker and Stainier, 2014). ...
Full-text available
Axonal growth and guidance at the ventral floor plate is here followed $\textit{in vivo}$ in real time at high resolution by light-sheet microscopy along several hundred micrometers of the zebrafish spinal cord. The recordings show the strikingly stereotyped spatio-temporal control that governs midline crossing. Commissural axons are observed crossing the ventral floor plate midline perpendicularly at about 20 microns/h, in a manner dependent on the Robo3 receptor and with a growth rate minimum around the midline, confirming previous observations. At guidance points, commissural axons are seen to decrease their growth rate and growth cones increase in size. Commissural filopodia appear to interact with the nascent neural network, and thereby trigger immediate plastic and reversible sinusoidal-shaped bending movements of neighboring commissural shafts. Ipsilateral axons extend concurrently, but straight and without bends, at three to six times higher growth rates than commissurals, indicating they project their path on a substrate-bound surface rather than relying on diffusible guidance cues. Growing axons appeared to be under stretch, an observation that is of relevance for tension-based models of cortical morphogenesis. The \textit{in vivo} observations provide for a discussion of the current distinction between substrate-bound and diffusible guidance cues. The study applies the transparent zebrafish model that provides an experimental model system to explore further the cellular, molecular and physical mechanisms involved during axonal growth, guidance and midline crossing through a combination of $\textit{in vitro}$ and $\textit{in vivo}$ approaches.
... Considering all the solutions available to breeders to improve pastures cultivars from a molecular perspective, in vitro culture and Agrobacterium transformation, in combination with other technologies such as genome-wide association studies [26] and CRISPR [27], play a central role in the association of genes with agronomic traits. In this regard, analysing genes associated with biotic and abiotic stresses can unleash the power of plant adaptation, creating better pastures adapted to a range of different environmental conditions. ...
Full-text available
Subterranean clover (Trifolium subterraneum) is the most widely grown annual pasture legume in southern Australia. With the advent of advanced sequencing and genome editing technologies, a simple and efficient gene transfer protocol mediated by Agrobacterium tumefaciens was developed to overcome the hurdle of genetic manipulation in subterranean clover. In vitro tissue culture and Agrobacterium transformation play a central role in testing the link between specific genes and agronomic traits. In this paper, we investigate a variety of factors affecting the transformation in subterranean clover to increase the transformation efficiency. In vitro culture was optimised by including cefotaxime during seed sterilisation and testing the best antibiotic concentration to select recombinant explants. The concentrations for the combination of antibiotics obtained were as follows: 40 mg L−1 hygromycin, 100 mg L−1 kanamycin and 200 mg L−1 cefotaxime. Additionally, 200 mg L−1 cefotaxime increased shoot regeneration by two-fold. Different plant hormone combinations were tested to analyse the best rooting media. Roots were obtained in a medium supplemented with 1.2 µM IAA. Plasmid pH35 containing a hygromycin-resistant gene and GUS gene was inoculated into the explants with Agrobacterium tumefaciens strain AGL0 for transformation. Overall, the transformation efficiency was improved from the 1% previously reported to 5.2%, tested at explant level with Cefotaxime showing a positive effect on shooting regeneration. Other variables in addition to antibiotic and hormone combinations such as bacterial OD, time of infection and incubation temperature may be further tested to enhance the transformation even more. This improved transformation study presents an opportunity to increase the feeding value, persistence, and nutritive value of the key Australian pasture.
... Table 1. Overview of CRISPR-Cas classification and characteristics [17][18][19][20]. All types are distinguished by different architectures of the effector modules, which contain unique signature proteins. ...
Full-text available
The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR–Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR–Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science. Although CRISPR–Cas9 allows easy genome editing and has a number of benefits, we should not ignore the important ethical and biosafety issues. Moreover, any tool that has great potential and offers significant capabilities carries a level of risk of being used for non-legal purposes. In this review, we present a brief history and mechanism of the CRISPR–Cas9 system. We also describe on the applications of this technology in gene regulation and genome editing; the treatment of cancer and other diseases; and limitations and concerns of the use of CRISPR–Cas9.
In industrial applications such as fermentation and heterologous protein production, various Aspergillus oryzae and A. sojae strains are used. Although genetic engineering techniques have been developed for these filamentous fungi, applying such classical techniques to many strains is difficult. Therefore, the establishment of innovative technologies applicable to various industrial strains is required. We previously developed a genome editing technology using the CRISPR/Cas9 system for the efficient genetic engineering of A. oryzae; however, this system is limited by its protospacer adjacent motif sequence. In A. sojae, no genetic engineering using genome editing has been developed. In this study, we aimed to develop a genome editing technology using the Cpf1 nuclease for the genetic engineering of A. oryzae and A. sojae. AMA1-based genome editing vectors bearing codon-optimized cpf1 expression cassettes were constructed, and guide RNA expression cassettes were inserted into the Cpf1 genome editing vectors. Using the resultant plasmids, we performed mutagenesis of the AowA and sC genes in A. oryzae and the AswA gene in A. sojae. We deleted these genes by co-introducing the Cpf1 genome editing plasmid and the donor plasmid. Our study demonstrates that the CRISPR/Cpf1 system can be used as an efficient alternative to the CRISPR/Cas9 system to genetically engineer A. oryzae and as a new approach for efficient genetic engineering of A. sojae.
The development of radioresistance by nasopharyngeal carcinoma (NPC) cells almost always results in tumor recurrence and metastasis, making clinical treatment of the disease difficult. In this study, the mechanism of radioresistance in NPC cells was investigated. First, a gene array and quantitative reverse-transcription-PCR assays were used to screen for genes exhibiting significantly altered expression in the DNA damage signaling pathway. Based on those results, GADD45G was further studied in the context of radioresistance. A GADD45G-knockout NPC cell line (CNE-2R-KO) was constructed using CRISPR-Cas9 technology and used for a comparison of differences in radioresistance with other radiosensitive and radioresistant NPC cells, as evaluated using colony formation assays. Cell cycle changes were observed using flow cytometry. Cell proliferation and migration were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and wound healing assays, respectively. The sequencing results revealed the successful construction of the CNE-2R-KO cell line, the radiosensitivity of which was higher than that of its parent radioresistant cell line owing to the GADD45G knockout. This was likely related to the increase in the number of cells in the G1 phase and decrease in those in the S1 phase as well as the increased cell proliferation rate and decreased migratory ability. GADD45G is associated with radioresistance in NPC cells and likely has a role in the occurrence and metastasis of NPC.
The discovery of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, and its development into a set of powerful tools for manipulating the genome, has revolutionized genome editing. Precise, targeted CRISPR/Cas-based genome editing has become the most widely used platform in organisms ranging from plants to animals. The CRISPR/Cas system has been extensively modified to increase its efficiency and fidelity. In addition, the fusion of various protein motifs to Cas effector proteins has facilitated diverse set of genetic manipulations, such as base editing, transposition, recombination, and epigenetic regulation. The CRISPR/Cas system is undergoing continuous development to overcome current limitations, including off-target effects, narrow targeting scope, and issues associated with the delivery of CRISPR components for genome engineering and therapeutic approaches. Here, we review recent progress in a diverse array of CRISPR/Cas-based tools. We also describe limitations and concerns related to the use of CRISPR/Cas technologies.
Full-text available
Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib's target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents.
Full-text available
RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs typically relies on the introduction of exogenous tags. Here we demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.
Full-text available
Objective: Functional investigation of novel gene/protein targets associated with adipocyte differentiation or function heavily relies on efficient and accessible tools to manipulate gene expression in adipocytes in vitro. Recent advances in gene-editing technologies such as CRISPR-Cas9 have not only eased gene editing but also greatly facilitated modulation of gene expression without altering the genome. Here, we aimed to develop and validate a competent in vitro adipocyte model of controllable functionality as well as multiplexed gene manipulation in adipocytes, using the CRISPRa “SAM” system and siRNAs to simultaneously overexpress and silence selected genes in the same cell populations. Methods: We introduced a stable expression of dCas9-VP64 and MS2-P65, the core components of the CRIPSRa SAM system, in mesenchymal C3H/10T1/2 cells through viral delivery and used guide RNAs targeting Pparγ2, Prdm16, Zfp423, or Ucp1 to control the expression of key genes involved in adipocyte differentiation and function. We additionally co-transfected mature adipocytes with sgRNA plasmids and siRNA to simultaneously up-regulate and silence selected genes. Quantitative gene expression, oxygen consumption, fluorescence-activated cell sorting and immunocytochemistry served as validation proxies in pre- or mature adipocytes. Results: CRISPRa SAM-mediated up-regulation of a key adipogenic gene, Pparγ2, was successfully achieved using selected sgRNAs targeting the Pparγ2 promoter region (i.e. up to 104 fold); this induction was long lasting and sufficient to promote adipogenesis. Furthermore, co-activation of Pparγ2 with either Prdm16 or Zfp423 transcripts drove distinct thermogenic gene expression patterns associated with increased or decreased oxygen consumption, respectively, mimicking typical characteristics of brite/beige or white cell lineages. Lastly, we demonstrated that up-regulation of endogenous genes in mature adipocytes was also easily and efficiently achieved using CRISPRa SAM, here exemplified by targeted Ucp1 overexpression (up to 4 × 103 fold), and that it was compatible with concomitant gene silencing using siRNA, allowing for bidirectional manipulation of gene expression in the same cell populations. Conclusions: We demonstrate that the CRISPRa SAM system can be easily adopted and used to efficiently manipulate gene expression in pre- and mature adipocytes in vitro. Moreover, we describe a novel methodological approach combining the activation of endogenous genes and siRNA-mediated gene silencing, thus providing a powerful tool to functionally decipher genetic factors controlling adipogenesis and adipocyte functions.
Full-text available
The discovery and adaption of bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems has revolutionized the way researchers edit genomes. Engineering of catalytically inactivated Cas variants (nuclease-deficient or nuclease-deactivated [dCas]) combined with transcriptional repressors, activators, or epigenetic modifiers enable sequence-specific regulation of gene expression and chromatin state. These CRISPR–Cas-based technologies have contributed to the rapid development of disease models and functional genomics screening approaches, which can facilitate genetic target identification and drug discovery. In this short review, we will cover recent advances of CRISPR–dCas9 systems and their use for transcriptional repression and activation, epigenome editing, and engineered synthetic circuits for complex control of the mammalian genome.
Full-text available
Selection-free genome editing using Cas9 ribonucleoprotein embryo bombardment has been achieved for maize and wheat. This is a breakthrough that should make new breeding technologies more acceptable for worldwide use.
Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided RNases Cas13. Here, we profile Type VI systems to engineer a Cas13 ortholog capable of robust knockdown and demonstrate RNA editing by using catalytically-inactive Cas13 (dCas13) to direct adenosine to inosine deaminase activity by ADAR2 to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineer this system to create a high specificity variant and minimize the system to facilitate viral delivery. REPAIR presents a promising RNA editing platform with broad applicability for research, therapeutics, and biotechnology.
Next-generation DNA sequencing technologies have led to a massive accumulation of genomic and transcriptomic data from patients and healthy individuals. The major challenge ahead is to understand the functional significance of the elements of the human genome and transcriptome, and implications for diagnosis and treatment. Genetic screens in mammalian cells are a powerful approach to systematically elucidate gene function in health and disease states. In particular, recently developed CRISPR/Cas9-based screening approaches have enormous potential to uncover mechanisms and therapeutic strategies for human diseases. The focus of this review is the use of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) for genetic screens in mammalian cells. We introduce the underlying technology and present different types of CRISPRi/a screens, including those based on cell survival/proliferation, sensitivity to drugs or toxins, fluorescent reporters, and single-cell transcriptomes. Combinatorial screens, in which large numbers of gene pairs are targeted to construct genetic interaction maps, reveal pathway relationships and protein complexes. We compare and contrast CRISPRi and CRISPRa with alternative technologies, including RNA interference (RNAi) and CRISPR nuclease-based screens. Finally, we highlight challenges and opportunities ahead.
With the rapid increase in the global population and the impact of climate change on agriculture, there is a need for crops with higher yields and greater tolerance to abiotic stress. However, traditional crop improvement via genetic recombination or random mutagenesis is a laborious process and cannot keep pace with increasing crop demand. Genome editing technologies such as clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) allow targeted modification of almost any crop genome sequence to generate novel variation and accelerate breeding efforts. We expect a gradual shift in crop improvement away from traditional breeding towards cycles of targeted genome editing. Crop improvement using genome editing is not constrained by limited existing variation or the requirement to select alleles over multiple breeding generations. However, current applications of crop genome editing are limited by the lack of complete reference genomes, the sparse knowledge of potential modification targets, and the unclear legal status of edited crops. We argue that overcoming technical and social barriers to the application of genome editing will allow this technology to produce a new generation of high-yielding, climate ready crops.
Variation in prokaryote adaptive immunity To repel infection by phage and mobile genetic elements, prokaryotes have a form of adaptive immune response and memory invested in clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas). This molecular machinery can recognize and remember foreign nucleic acids by capturing and retaining small nucleotide sequences. On subsequent encounters, the cognate CRISPR-Cas marshals enzymatic defenses to destroy infecting elements that contain the same sequences. Jackson et al. review the molecular mechanisms by which diverse CRISPR-Cas systems adapt and anticipate novel threats and evasive countermeasures from mobile genetic elements. Science , this issue p. eaal5056