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A high level of specificity is achieved with ZFNs because the Fok1 must dimerize for cleav-
age to occur. As such, a pair of ZFNs must be designed (one each side of the cut site on opposite
strands) and only when dual binding occurs does dimerization and cleavage ensue. Once the cut has
been achieved, the cell activates repair pathways to fix the damage. The non-homologous end join-
ing (NHEJ) pathway is error-prone, and this can be particularly useful to introduce disruptions in
the coding region of a gene. Small insertions and deletions are commonly introduced at
the cut site,
often resulting in disruption of the coding frame of an associated gene. ZFNs are therefore particu-
larly effective at knocking out gene function.
A modification of this approach is the use of transcription activator-like effector nucleases
(TALENs). TALENs are made by fusing a group of DNA-binding domains to a DNA cleavage do-
main. Transcription activator-like effectors (TALEs) can be engineered to bind to practically any
desired
DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations.
Studying the acquired immunity in bacteria yet another nuclease was identified that ap-
peared capable of cutting at precise location. As these nucleases were initially found to be associat-
ed to some Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) they were
named Cas (CRISPR-associated) proteins. Several such proteins were identified (of which Cas-9 is
the most widely utilized) the activity of which was later found to be triggered by the presence of a
specific small RNAs, called small guiding RNAs (sgRNAs). As these RNAs are quite short creating
them appeared very simple and this made this entire system very easy to use. Another very useful
property of the system is that the sgRNA can bind on either strand of DNA and the Cas9 will cleave
both strands (double strand break, DSB). Such DSB results in the silencing of that DNA sequence
and allows for either removing or inserting specific nucleotides at the site of cleavage. Changing
the target specificity of the RNA-protein complex does not require protein engineering but only the
design of the short crRNA guide.
Figure. Genomic manipulation with the CRISPR-Cas system requires only the Cas9
protein
and an engineered small guide RNA (sgRNA) with a protospacer associated motif (PAM)
sequence upstream of target complementary sequence.
Owing to this ease of customization, CRISPR–Cas9 is now used to edit or modify the ge-
nomes of a vast number of cells and organisms, including bacteria, parasites, plants, zebrafish, mice
and human cells. When co-expressed with custom-designed sgRNAs in cells, Cas9
generates double
strand brakes (DSBs) in genomic DNA that are subsequently repaired by non-homologous end-
joining (NHEJ) to introduce gene disruptions or by homology directed repair (HDR) through the
insertion of donor genetic sequences. Introducing DSBs at defined positions can generate cell lines
carrying chromosomal translocations that resemble those that occur in naturally observed cell line-
ages. In addition, the targeting of several loci simultaneously with multiple sgRNAs has also been
achieved, which is known as multiplexing. CRISPR–Cas9 thus provides a robust and malleable tool
to study genomic rearrangements, and increases our understanding of the development of different
tolerance mechanisms.
A modification of this system uses novel non-Cas9 CRISPR system isolated from
Francisel-
la novicida strain. It differs from Cas9 in that:
It prefers a protospacer associated motif (PAM) that is located 5' to the cut site,
The RNA sequence required for genome editing is significantly shorter at about
43 nucleotides long (Cas9 by comparison needs about 100nt),
Cleavage by Cpf1 results in 'sticky ends' due to cut sites being staggered by about
5 bases.
The discovery of this system further expands the genome editing tool box – potentially mak-
ing available target sites that, due to PAM constraints, would not be available to Cas9. Further to
this the nature of Cpf1 cuts, being both sticky and distal to the PAM site, may have some added ad-
vantages for homology directed repair.
Gene regulation
A key property of Cas9 is its ability to bind to DNA at sites defined by the guide RNA se-
quence and the PAM, which enables applications beyond permanent modification of DNA. In par-
ticular, dCas9 in combination with engineered sgRNAs has been repurposed for targeted gene regu-
lation on a genome-wide scale. In a process known as CRISPRi, dCas9 can be used to block RNA
polymerase‘s access to the DNA, and thereby reversibly repress transcription in bacteria, plant and
in human cells. This is usually achieved by employing catalytically inactive version of Cas9 (with-
out endonucleolytic activity). As the sgRNA-Cas
complex can bind to regions, involved in gene
regulation, modified transcription can be achieved without genetic alteration of the target gene se-
quence. On the other hand premature termination of translation can be achieved by using sgRNA
comprising of 3 segments: 20-nt sequence complementary to target DNA, 42-nt Cas9-binding hair-
pin (Cas9 handle), and 40-nt transcription terminator. The flexibility of the CRISPR-Cas
system can
be used in the opposite direction as well. Instead of blocking/reducing gene expression it can result
in efficient gene activation. This can be achieved by generating chimeric versions of dCas9 that are
fused to regulatory domains (such as the RNA polymerase ω-subunit). These variable uses of the
system are united under the term CRISPR interference (CRISPRi).
All these possibilities open up the doors to fine tuning gene expression and therefore – bet-
ter understanding and regulating the key players in plant heavy metal tolerance pathways as re-
quired for their economically viable implementation in crops.
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