The development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a long-standing goal
for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus
pyogenes has generated considerable excitement (1). This follows several attempts over the years to manipulate gene function, including
homologous recombination (2) and RNA interference (RNAi) (3). RNAi, in particular, became a laboratory staple enabling inexpensive
and high-throughput interrogation of gene function (4, 5), but it is hampered by providing only temporary inhibition of gene function
and unpredictable off-target effects (6). Other recent approaches to targeted genome modification – zinc-finger nucleases [ZFNs, (7)] and
transcription-activator like effector nucleases [TALENs (8)]– enable researchers to generate permanent mutations by introducing doublestranded
breaks to activate repair pathways. These approaches are costly and time-consuming to engineer, limiting their widespread use,
particularly for large scale, high-throughput studies.
The Biology of Cas9
The functions of CRISPR (Clustered Regularly
Interspaced Short Palindromic Repeats) and
CRISPR-associated (Cas) genes are essential in
adaptive immunity in select bacteria and archaea,
enabling the organisms to respond to and
eliminate invading genetic material. These repeats
were initially discovered in the 1980s in
E. coli (9), but their function wasn’t confirmed
until 2007 by Barrangou and colleagues, who
demonstrated that S. thermophilus can acquire resistance
against a bacteriophage by integrating
a genome fragment of an infectious virus into its
CRISPR locus (10).
Three types of CRISPR mechanisms have been
identified, of which type II is the most studied. In
this case, invading DNA from viruses or plasmids
is cut into small fragments and incorporated into
a CRISPR locus amidst a series of short repeats
(around 20 bps). The loci are transcribed, and
transcripts are then processed to generate small
RNAs (crRNA – CRISPR RNA), which are used to guide effector endonucleases that target
invading DNA based on sequence complementarity
(Figure 1) (11).
One Cas protein, Cas9 (also known as Csn1),
has been shown, through knockdown and rescue
experiments to be a key player in certain CRISPR
mechanisms (specifically type II CRISPR systems).
The type II CRISPR mechanism is unique compared
to other CRISPR systems, as only one Cas protein
(Cas9) is required for gene silencing (12). In type
II systems, Cas9 participates in the processing of
crRNAs (12), and is responsible for the destruction of
the target DNA (11). Cas9’s function in both of these
steps relies on the presence of two nuclease domains,
a RuvC-like nuclease domain located at the amino
terminus and a HNH-like nuclease domain that resides
in the mid-region of the protein (13).
To achieve site-specific DNA recognition and
cleavage, Cas9 must be complexed with both a
crRNA and a separate trans-activating crRNA
(tracrRNA or trRNA), that is partially complementary
to the crRNA (11). The tracrRNA is required
for crRNA maturation from a primary transcript
encoding multiple pre-crRNAs. This occurs in the
presence of RNase III and Cas9 (12).
During the destruction of target DNA, the HNH
and RuvC-like nuclease domains cut both DNA
strands, generating double-stranded breaks (DSBs)
at sites defined by a 20-nucleotide target sequence
within an associated crRNA transcript (11, 14).
The HNH domain cleaves the complementary
strand, while the RuvC domain cleaves the noncomplementary
The double-stranded endonuclease activity of Cas9
also requires that a short conserved sequence, (2–5
nts) known as protospacer-associated motif (PAM),
follows immediately 3´- of the crRNA complementary
sequence (15). In fact, even fully complementary
sequences are ignored by Cas9-RNA in the
absence of a PAM sequence (16).
Cas9 and CRISPR as a New Tool in
The simplicity of the type II CRISPR nuclease,
with only three required components (Cas9 along
with the crRNA and trRNA) makes this system
amenable to adaptation for genome editing. This
potential was realized in 2012 by the Doudna
and Charpentier labs (11). Based on the type II
CRISPR system described previously, the authors
developed a simplified two-component system
by combining trRNA and crRNA into a single
synthetic single guide RNA (sgRNA). sgRNAprogrammed
Cas9 was shown to be as effective
as Cas9 programmed with separate trRNA and
crRNA in guiding targeted gene alterations
To date, three different variants of the Cas9
nuclease have been adopted in genome-editing
protocols. The first is wild-type Cas9, which
can site-specifically cleave double-stranded
DNA, resulting in the activation of the doublestrand
break (DSB) repair machinery. DSBs can
be repaired by the cellular Non-Homologous
End Joining (NHEJ) pathway (17), resulting
in insertions and/or deletions (indels) which
disrupt the targeted locus. Alternatively, if a donor template with homology to the targeted
locus is supplied, the DSB may be repaired by
the homology-directed repair (HDR) pathway
allowing for precise replacement mutations to be
made (Figure 2A) (17, 18).
Cong and colleagues (1) took the Cas9 system
a step further towards increased precision by
developing a mutant form, known as Cas9D10A,
with only nickase activity. This means it cleaves
only one DNA strand, and does not activate
NHEJ. Instead, when provided with a homologous
repair template, DNA repairs are conducted via
the high-fidelity HDR pathway only, resulting in
reduced indel mutations (1, 11, 19). Cas9D10A is
even more appealing in terms of target specificity
when loci are targeted by paired Cas9 complexes
designed to generate adjacent DNA nicks (20) (see
further details about “paired nickases” in Figure 2B).
The third variant is a nuclease-deficient Cas9
(dCas9, Figure 2C) (21). Mutations H840A in the
HNH domain and D10A in the RuvC domain
inactivate cleavage activity, but do not prevent
DNA binding (11, 22). Therefore, this variant
can be used to sequence-specifically target any
region of the genome without cleavage. Instead, by fusing with various effector domains, dCas9
can be used either as a gene silencing or activation
tool (21, 23–26). Furthermore, it can be used
as a visualization tool. For instance, Chen and
colleagues used dCas9 fused to Enhanced Green
Fluorescent Protein (EGFP) to visualize repetitive
DNA sequences with a single sgRNA or nonrepetitive
loci using multiple sgRNAs (27).
Targeting Efficiency and Off-target
Targeting efficiency, or the percentage of desired
mutation achieved, is one of the most important
parameters by which to assess a genome-editing
tool. The targeting efficiency of Cas9 compares
favorably with more established methods, such
as TALENs or ZFNs (8). For example, in human
cells, custom-designed ZFNs and TALENs could
only achieve efficiencies ranging from 1% to
50% (29–31). In contrast, the Cas9 system has
been reported to have efficiencies up to >70%
in zebrafish (32) and plants (33), and ranging
from 2–5% in induced pluripotent stem cells (34).
In addition, Zhou and colleagues were able to
improve genome targeting up to 78% in one-cell
mouse embryos, and achieved effective germline
transmission through the use of dual sgRNAs to
simultaneously target an individual gene (35).
A widely used method to identify mutations is the
T7 Endonuclease I mutation detection assay (36,
37) (Figure 3). This assay detects heteroduplex
DNA that results from the annealing of a DNA
strand, including desired mutations, with a wildtype
DNA strand (37).
Another important parameter is the incidence of
off-target mutations. Such mutations are likely to
appear in sites that have differences of only a few
nucleotides compared to the original sequence,
as long as they are adjacent to a PAM sequence.
This occurs as Cas9 can tolerate up to 5 base
mismatches within the protospacer region (36) or
a single base difference in the PAM sequence (38).
Off-target mutations are generally more difficult to
detect, requiring whole-genome sequencing to rule
them out completely.
Recent improvements to the CRISPR system for
reducing off-target mutations have been made
through the use of truncated gRNA (truncated
within the crRNA-derived sequence) or by adding
two extra guanine (G) nucleotides to the 5´ end
(28, 37). Another way researchers have attempted
to minimize off-target effects is with the use of
“paired nickases” (20). This strategy uses D10A
Cas9 and two sgRNAs complementary to the
adjacent area on opposite strands of the target site
(Figure 2B). While this induces DSBs in
the target DNA, it is expected to create only single nicks in off-target locations and, therefore, result
in minimal off-target mutations.
By leveraging computation to reduce off-target
mutations, several groups have developed webbased
tools to facilitate the identification of
potential CRISPR target sites and assess their
potential for off-target cleavage. Examples
include the CRISPR Design Tool (38) and the
ZiFiT Targeter, Version 4.2 (39, 40).
Applications as a Genome-editing
and Genome Targeting Tool
Following its initial demonstration in 2012
(9), the CRISPR/Cas9 system has been widely
adopted. This has already been successfully used
to target important genes in many cell lines and
organisms, including human (34), bacteria (41),
zebrafish (32), C. elegans (42), plants (34), Xenopus
tropicalis (43), yeast (44), Drosophila (45), monkeys
(46), rabbits (47), pigs (42), rats (48) and mice
(49). Several groups have now taken advantage of
this method to introduce single point mutations
(deletions or insertions) in a particular target
gene, via a single gRNA (14, 21, 29). Using a
pair of gRNA-directed Cas9 nucleases instead,
it is also possible to induce large deletions or
genomic rearrangements, such as inversions
or translocations (50). A recent exciting
development is the use of the dCas9 version
of the CRISPR/Cas9 system to target protein
domains for transcriptional regulation (26, 51,
52), epigenetic modification (25), and microscopic
visualization of specific genome loci (27).
The CRISPR/Cas9 system requires only the redesign
of the crRNA to change target specificity.
This contrasts with other genome editing tools,
including zinc finger and TALENs, where redesign
of the protein-DNA interface is required.
Furthermore, CRISPR/Cas9 enables rapid
genome-wide interrogation of gene function
by generating large gRNA libraries (51, 53) for
The future of CRISPR/Cas9
The rapid progress in developing Cas9 into a set
of tools for cell and molecular biology research
has been remarkable, likely due to the simplicity,
high efficiency and versatility of the system. Of the
designer nuclease systems currently available for
precision genome engineering, the CRISPR/Cas
system is by far the most user friendly. It is now
also clear that Cas9’s potential reaches beyond
DNA cleavage, and its usefulness for genome
locus-specific recruitment of proteins will likely
only be limited by our imagination.
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From NEB expressions Issue I, 2014
Article by Alex Reis, Ph.D., Bitesize Bio
Breton Hornblower, Ph.D., Brett Robb, Ph.D. and George Tzertzinis, Ph.D., New England