interspaced short palindromic repeats- (CRISPR-) CRISPR associated protein 9 (Cas9) system. ZFNs were directly discarded as
they remain difficult to engineer and prone to failure [75]. TALENs
were originally derived from the TAL effector proteins of tomato
pathogen Xanthomonas, and reduced transformation efficiencies
have raised concerns about potential TALEN cytotoxicity [76]. In
contrast, CRISPR-Cas9 has been repeatedly used in plants with
virtually none of the presented drawbacks [77, 78].
To create different mutant alleles of a gene, we must first design
guide RNAs (gRNAs). The gRNAs are aimed to recognize a DNA
sequence in the genome containing a 3’ protospacer adjacent motif
(PAM), which would ultimately be targeted by the Cas9. Cleavage
occurs around 3 bp upstream of the PAM domain, and those DNA
breaks will eventually be repaired with deletions of nucleotides. In
order to design spacer gRNA sequences specific to our gene of
interest:
1. Go to https://www.genome.arizona.edu/crispr/CRISPRsearch.
html. In the search box, search for “AT3G24650” and select
“Arabidopsis thaliana” as the species.
2. The output will contain potential spacer sequences to be used
for the gRNAs. These sequences are classified based on their
potential off-target effects (Class0.0 being the lowest of all).
The table also contains valuable information such as the exact
genomic position of the targeting, which will be necessary for
screening for mutations in plants exposed to these CRISPRgRNAs.
3. Select a gRNA sequence by clicking on the empty box at the
end of the row. This will expand the table and offer information
about the cloning of this gRNA sequence, and whether it is
possible to use restriction enzymes for cut analysis during
genotyping. For cloning, a published plasmid vector (pRGE),
containing necessary components to express Cas9 and gRNA,
is suggested, and several other compatible alternatives exist.
3.12.2 Artificial miRNA
In certain cases, obtaining loss-of-function mutation for functional
analysis of a gene is not a viable approach. Full knockouts might be
lethal or we may need a more specific spatiotemporal reduction of
the activity of our gene of interest. In such scenarios, the use of an
artificial miRNA will be a valuable solution. The artificial microRNA (amiRNA) technology exploits endogenous miRNA precursors to generate sRNAs that direct gene silencing [79]. These
amiRNAs are 21nt small RNAs that are genetically engineered to
target and silence single or multiple genes [80]. The level and
spatiotemporal characteristics of the silencing ultimately depend
on the expression profile of the amiRNA, allowing for more flexible
frameworks for investigating gene function. The Web MicroRNA
Arabidopsis Bioinformatics
79
they remain difficult to engineer and prone to failure [75]. TALENs
were originally derived from the TAL effector proteins of tomato
pathogen Xanthomonas, and reduced transformation efficiencies
have raised concerns about potential TALEN cytotoxicity [76]. In
contrast, CRISPR-Cas9 has been repeatedly used in plants with
virtually none of the presented drawbacks [77, 78].
To create different mutant alleles of a gene, we must first design
guide RNAs (gRNAs). The gRNAs are aimed to recognize a DNA
sequence in the genome containing a 3’ protospacer adjacent motif
(PAM), which would ultimately be targeted by the Cas9. Cleavage
occurs around 3 bp upstream of the PAM domain, and those DNA
breaks will eventually be repaired with deletions of nucleotides. In
order to design spacer gRNA sequences specific to our gene of
interest:
1. Go to https://www.genome.arizona.edu/crispr/CRISPRsearch.
html. In the search box, search for “AT3G24650” and select
“Arabidopsis thaliana” as the species.
2. The output will contain potential spacer sequences to be used
for the gRNAs. These sequences are classified based on their
potential off-target effects (Class0.0 being the lowest of all).
The table also contains valuable information such as the exact
genomic position of the targeting, which will be necessary for
screening for mutations in plants exposed to these CRISPRgRNAs.
3. Select a gRNA sequence by clicking on the empty box at the
end of the row. This will expand the table and offer information
about the cloning of this gRNA sequence, and whether it is
possible to use restriction enzymes for cut analysis during
genotyping. For cloning, a published plasmid vector (pRGE),
containing necessary components to express Cas9 and gRNA,
is suggested, and several other compatible alternatives exist.
3.12.2 Artificial miRNA
In certain cases, obtaining loss-of-function mutation for functional
analysis of a gene is not a viable approach. Full knockouts might be
lethal or we may need a more specific spatiotemporal reduction of
the activity of our gene of interest. In such scenarios, the use of an
artificial miRNA will be a valuable solution. The artificial microRNA (amiRNA) technology exploits endogenous miRNA precursors to generate sRNAs that direct gene silencing [79]. These
amiRNAs are 21nt small RNAs that are genetically engineered to
target and silence single or multiple genes [80]. The level and
spatiotemporal characteristics of the silencing ultimately depend
on the expression profile of the amiRNA, allowing for more flexible
frameworks for investigating gene function. The Web MicroRNA
Arabidopsis Bioinformatics
79