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

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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.
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Bioengineered
ISSN: 2165-5979 (Print) 2165-5987 (Online) Journal homepage: http://www.tandfonline.com/loi/kbie20
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:
10.1080/21655979.2018.1470720
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COMMENTARY
CRISPR-Cas systems: ushering in the new genome editing era
Fernando Perez Rojo
a
, Rikard Karl Martin Nyman
a
, Alexander Arthur Theodore Johnson
b
,
Maria Pazos Navarro
a,c
, Megan Helen Ryan
c,d
, William Erskine
a,c
, and Parwinder Kaur
a,c,e
a
Centre for Plant Genetics and Breeding, School of Agriculture and Environment, The University of Western Australia, Crawley, WA, Australia;
b
School of BioSciences, The University of Melbourne, Victoria, Australia;
c
Institute of Agriculture, The University of Western Australia, Crawley,
WA, Australia;
d
School of Agriculture and Environment, The University of Western Australia, Crawley, WA, Australia;
e
Telethon Kids Institute,
Subiaco, WA, Australia
ABSTRACT
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.
ARTICLE HISTORY
Accepted 25 April 2018
KEYWORDS
CRISPR products; Cas9;
Cas13; genome editing;
NHEJ; HDR; ZFN; TALEN;
CRISPR-Cas figure; Cas
classes; Cas types
Introduction
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
bluntendsofthecleavedtargetDNAproductatthe
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 parwinder.kaur@uwa.edu.au Centre for Plant Genetics and Breeding, School of Agriculture and Environment, The
University of Western Australia, Crawley, WA 6009, Australia.
BIOENGINEERED
2018, VOL. 9, NO. 1, 214221
https://doi.org/10.1080/21655979.2018.1470720
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.
BIOENGINEERED 215
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
Integration
module
Cas1/2 [3] Cas1/2 Unknown Cas1/2 Cas1/2 Cas1/2
Effector
module
47 Cas protein Cascade Cas9 [10] Cas12a (cpf1)/Cas12b/
Cas12c/[17]
Cas13a/Cas13b/
Cas13c [20]
Molecule
substrate
DNA RNA
Organism bacteria and archaea archaea bacteria bacteria and archaea bacteria
Nuclease
domain
HD
a
fused to
Cas3
HD fused to
Cas10
unknown RuvC and HNH RuvC and Nuc HEPN domains (2)
tracrRNA no No no yes cpf1-no no
Cleavage motif subtype
dependant (7)
subtype
dependant (2)
subtype
dependant (2)
CG rich NGG (blunt
ends)
AT rich (staggered ends) non-G PFS (ssRNA)
a
Histidine-aspartate domain
216 F. PEREZ ROJO ET AL.
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
markers
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
BIOENGINEERED 217
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.
218 F. PEREZ ROJO ET AL.
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. Conclusion 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. Acknowledgments 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. 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Available from: http://www.sciencemag. org.ezproxy.library.uwa.edu.au/news/2017/07/ding- ding-ding-crispr-patent-fight-enters-next-round [38] CRISPR COMMERCIALIZATION RISK | REGENHEALTHSOLUTIONS [Internet]. [cited 2017 Dec 29]. Available from: http://www.regenhealthsolu tions.info/4/crispr_commercialization_risk_780838.html [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. BIOENGINEERED 221 ... 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]. ... Article 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). ... Article 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. ... Article 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? ... Article 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). ... Preprint 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. ...
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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. ...
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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