CRISPR 101

Your Guide to Understanding CRISPR

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Introduction to Genome Editing

Genome editing involves the deletion, insertion, or modification of
the genome at a specific site in a DNA sequence. For many years,
researchers had been trying to develop easy and cost-effective
genome editing tools to address problems across a wide spectrum of
fields. For instance, gene therapy in humans could progress rapidly if
one could simply eliminate the gene responsible for a certain genetic
disorder. In agriculture, manipulating plant DNA could be used to
optimize crop yields and control plant diseases. Similarly, bacterial
genomes could be fine-tuned to increase their product yields in
several industrial applications.

Finally, the efforts of researchers paid off with the development

of CRISPR, a robust molecular tool that can edit DNA at virtually
any locus. CRISPR technology is igniting a revolution across the life
sciences and is quickly becoming a standard tool in many labs. Given
its ease-of-use and versatility, CRISPR is already used for a variety of
applications and holds a lot of promise for the future.

Read on for a crash course in everything you need to know about the
fundamentals of CRISPR.

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Genome Editing Tools Before CRISPR

Although CRISPR has now become synonymous with gene editing, it is of sequences. Although they are easier to design than ZFNs, TALENs are
not the first technology developed to edit DNA. Rather, the pioneers of expensive to produce.
the genome engineering field were zinc-finger nucleases (ZFNs).
An additional genome editing technique uses engineered restriction
The ZFN method involves engineering an enzyme with both a zinc finger enzymes in concert with recombinant adeno-associated viruses (rAAVs).
DNA-binding domain and a restriction endonuclease domain. The zinc AAV is a non-pathogenic virus that infects mammalian cells at all stages
finger domain is designed to target and bind to specific sequences of of the cell cycle and integrates into the host genome at predictable sites.
DNA, and the nuclease domain cleaves the DNA at the desired site. The AAV genome can be modified to target specific sequences in the host
Although ZFN editing represented the first breakthrough in site-specific genome and integrate desired modifications. However, the AAV approach
genome engineering, they have several limitations. In addition to also has several limitations. For instance, the vectors are difficult to
exhibiting off-target effects, ZFNs are expensive and time-consuming to produce and can only accommodate a small amount of genetic material.
engineer. Furthermore, their inefficiency limits their practical application
to only one genomic edit at a time. Up until now, the field of genome engineering has provided researchers
with a few gene editing technologies, all of which have limitations (Table
Many years after ZFNs made their debut, a similar method known as 1). Because ZFNs and TALENS require complex protein-DNA interactions,
transcription activator-like effector nucleases (TALENs) was developed. they are challenging design and manipulate. AAV vectors are also difficult
The TALENs method utilizes engineered enzymes containing a DNA- to work with and have limited applications given their small packaging
binding domain and a separate DNA-cleaving domain, similar to the ZFNs capacity. CRISPR, which relies on well-understood interactions between
method. However, TALENs have an advantage over ZFNs because they DNA and RNA, has offered a far simpler way of editing genes and has
are more flexible: their DNA-binding domains can target a wider range completely changed the face of genomic engineering.

Table 1. Comparison of gene editing technologies

T YP E ZFNs TALENs AAV CRISPR

COST High High Moderate Low

C OM P L E X I T Y Difficult Difficult Difficult Easy

MU L T I P L E EDI T S Difficult Difficult Difficult Easy

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History of CRISPR
The foundational discoveries that led to the development of CRISPR-
Cas9 technology can be traced back to 1993, when repetitive palindromic
segments of DNA interspaced with other fragments of genetic material
were identified in prokaryotes. These pieces of genetic code were named
Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR.

In 2007, after years of studying CRISPR genetic motifs, researchers

concluded that CRISPR’s function is related to immunity. It took combined
efforts of several research groups over the next 5 years to elucidate the
underlying molecular mechanism behind the CRISPR system.

As it turns out, bacteria and archaea use the CRISPR-Cas9 system to

defend themselves against invading viruses (called bacteriophages).
Upon encountering a viral infection, the prokaryotic cell employs a
special CRISPR-associated nuclease (Cas9) to snip-off a piece of viral DNA
by creating a double-strand break (DSB) in its target loci.

How does the Cas9 protein recognize the target DNA? It is directed to the
target sequence by a short RNA fragment known as a guide RNA (gRNA).
The guide RNA is complementary to a segment of the viral genome,
CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats. A set of DNA
which allows Cas9 to cleave DNA with a high degree of specificity.
sequences involved in the prokaryotic immune system and has been adapted for
genome engineering.
Not only does that destroy the virus, but the fragment of foreign DNA,
called a "spacer," may be stored between the palindromic sequences
of the CRISPR array as a way of retaining a genetic memory of past Once scientists figured out the mechanism of CRISPR in prokaryotes, it
infections. If the virus were to re-invade, the CRISPR-Cas9 system could did not take long for them to realize the potential for engineering the
quickly target and destroy it. This library of viral fragments is thus genomes of microbes, plants, and animals. Today, CRISPR is utilized
essentially equivalent to our immune system, which stores antigens to for a variety of applications and its adoption continues to increase in
prepare for future infections. laboratories throughout the world.

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Cas9
The Components of CRISPR
The CRISPR system comprises two components: a guide RNA (gRNA)
that is specific to the target DNA sequence and a non-specific CRISPR- gRNA
associated endonuclease protein (Cas9).

5' 3'

N
CC
The Cas9 protein functions as a pair of molecular scissors, while the 3' 5'

G
G
N
Target DNA PAM
gRNA is the GPS that guides it to the appropriate site. In bacteria, the
gRNA guides nuclease to viral DNA, but as a biotechnological tool, the
design specifications of the gRNA can be altered to target the nuclease to
cleave any host organism’s genome at virtually any location.

Recognition of the target DNA by the Cas9 enzyme is subject to the

Figure 1. The CRISPR-Cas9 system. The CRISPR-Cas9 system comprises a guide RNA
presence of a short protospacer adjacent motif (PAM) sequence
(gRNA) and Cas9 nuclease, which together form a ribonucleoprotein (RNP) complex. The
located directly downstream on the untargeted DNA strand (Fig 1). If a presence of a specific protospacer adjacent motif (PAM) in the genomic DNA is required
for the gRNA to bind to the target sequence. The Cas9 nuclease then makes a double
correct match is made, Cas9 cleaves both DNA strands 3-4 nucleotides
strand break in the DNA (denoted by the scissors). Endogenous repair mechanisms
upstream of the PAM site. In nature, this short genetic element only triggered by the double strand break may result in gene knockout via a frameshift
occurs in invading viruses (not the bacterial genome), and thus ensures mutation or knock-in of a desired sequence if a DNA template is present.

that Cas9 does not cleave its own CRISPR locus. The PAM sequence varies
for Cas9 proteins from different species: the PAM for the most widely sequences complementary to viral DNA. When used for gene editing,
used Cas9 from Streptococcus pyogenes is 5′-NGG-3′, where N is any however, the crRNA can be programmed to target virtually any genetic
nucleotide. sequence. The tracrRNA functions as a scaffold for the crRNA-Cas9
interaction. Guide RNAs naturally form a duplex molecule, with the
In its natural form, the gRNA consists of two distinct segments of crRNA and tracrRNA segments annealed together (crRNA:tracrRNA).
RNA: CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA). Synthetically, they can be engineered as one seamless fusion sequence
The crRNA is complementary to the target DNA sequence, and thus called a single guide RNA (sgRNA) (Fig 2).
recognizes the sequence to be cleaved. In nature, the RNA consists of

Guide RNA (gRNA): the RNA component of the CRISPR- CRISPR-associated endonuclease protein (Cas9): PAM (protospacer adjacent motif): a short sequence of
Cas9 genome engineering tool. Contains a guide sequence a common endonuclease used in the CRISPR-Cas9 genome nucleotides that must be present downstream of the target
that is complimentary to the genomic target. engineering tool. Makes a double strand break on the DNA site in order for the nuclease to make a double strand
` at the targeted genomic region. break.

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1. Components of CRISPR-Cas9 2. RNP Formation

Cas9 protein and guide RNA, either single guide RNA (sgRNA) or crRNA:tracrRNA. Cas9 and sgRNA form a ribonucleoprotein (RNP).

Linker Loop Cas9

3' 5'

Cas9 sgRNA tracrRNA crRNA:tracrRNA tracrRNA

OR
sgRNA RNP
~20 nt crRNA ~20 nt crRNA
5' 5'
3' 3'

gRNA Target Sequence gRNA Target Sequence

3. Cas9-Mediated Double-Strand Break 4a. NHEJ Repair

Cas9 cleaves targeted DNA sequence and causes a double-strand break (DSB). Non-homologous repair pathway that leads to an insertion or
deletion of nucleotides, potentially resulting in a gene knockout.

5' 3'
Cas9 3' 5'

Insertion

DSB
OR
sgRNA 5' 3'
3' 5' 5' 3'

5' 3' 3' 5'

N
CC

3' 5' Deletion

G
G
N

Target DNA PAM

4b. Homology-Directed Repair

Addition of a donor DNA repair template with a new sequence
flanked by homology arms leads to a gene knock-in.

Homology Arms

Figure 2. CRISPR-Cas9 genome editing. 1) The components of CRISPR are a guide RNA (either sgRNA or 5' 3'
Donor DNA
cr:tracr) and a Cas9 endonuclease. 2) The guide RNA and Cas9 form a ribonucleoprotein (RNP). 3) Inside Template
3' 5'
the cell, the RNP creates a double-strand break (DSB) at the genomic target. One of two endogenous
Knock-In Sequence
repair mechanisms may mend the break. 4a) Non-homologous end joining (NHEJ) repair) is error-prone
pathway that often inserts or deletes nucleotides (indels). If an indel causes a frameshift mutation, then
the target gene may lose function (knockout). 4b) If a DNA template is provided, then the homology-
directed repair (HDR) pathway may mend the break through homologous recombination. This pathway 5' 3'

can be used to knock in a desired sequence of DNA. 3' 5'

Knock-In Sequence

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Repairing CRISPR-Induced Breaks 5'

DSB
3'
3' 5'

The magic of CRISPR is in its ability to cleave both strands of DNA. Cells
must repair DSBs, or risk dying. Thus, all of the editing that comes from
CRISPR is due to the cell’s innate ability to repair itself. There are two 5' 3'

kinds of repair pathways, each of which can be exploited to make desired 3' 5'

Insertion
edits.
OR

Non-Homologous End Joining 5' 3'

3' 5'

If the objective of an experiment is to permanently disrupt a gene so that Deletion

no functional protein is made (knockout), then one can exploit the cell’s
non-homologous end joining (NHEJ) repair mechanism. NHEJ binds the Figure 3. Non-homologous end joining repair (NHEJ): an error-
prone cellular mechanism that repairs double strand breaks in DNA,
double stranded break back together, but it is prone to error and may but often inserts or deletes nucleotides (indels) in the process. Can
insert or delete nucleotides (called indels) in the process. If the number be used to induce knockouts.

of nucleotides inserted or deleted is not divisible by three, then it will

induce a frameshift mutation and likely terminate the gene’s function (Fig
DSB
3).
5' 3'
3' 5'

Homology-Directed Repair

Alternatively, if the objective of the experiment is to replace the targeted Homology arms

genetic element with a different sequence (e.g., knock-in), the cell can Repair
5' 3'
template
be directed towards an alternative repair pathway, homology-directed 3' 5'

Knockin sequence

repair (HDR). To accomplish this, a homologous DNA template bearing

the desired sequence must be introduced in the cell, along with the
CRISPR components. A certain number of cells will use this template to 5' 3'
3' 5'
repair the broken sequence via homologous recombination, thereby Knockin sequence

incorporating the desired edits into the genome (Fig 4).

Figure 4. Homology-directed repair (HDR): a cellular mechanism

Indel: the insertion or deletion of nucleotides in the genome. Often caused by NHEJ
that repairs double strand breaks in DNA by using a homologous
repair following a double strand break in DNA.
DNA template. Can be used to induce knock-ins.

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What Can We
Achieve Using
CRISPR?
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CRISPRi

The field of genome engineering expanded quickly CRISPRa Knock Out

once scientists realized the potential of CRISPR.

While mice have traditionally been the most popular
model organism for transgenic experiments, CRISPR
applications have also been demonstrated in a wide
Anti- CRISPR
range of cells and organisms, including human embryos. CRISPR Screen
In Figure 5, we have summarized the current techniques
that use CRISPR-Cas9 technology.

Gene
Knock-in
Silencing

Figure 5. Current techniques using CRISPR technology

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a knock-in (KI). CRISPR KI applications are achieved by inducing cells to

Knockouts
repair breaks in DNA through homology-directed repair (HDR). For HDR
The process of making a gene permanently inoperative (e.g., does not experiments, a DNA template containing the knock-in sequence flanked
encode functional protein) is called a knockout (KO). CRISPR’s ability by regions of homology must be introduced into cells along with the
to disrupt gene function relies on the error-prone nature of the NHEJ CRISPR components. During HDR, the template is used to precisely repair
mechanism. As described above, indels that cause shifts in the reading the severed target sequence, incorporating the knock-in sequence in the
frame of a gene will likely terminate the gene’s function. This is especially process. HDR enables countless genomic re-writing applications, from
true for frameshifts that cause premature stop codons. By purposefully introducing single point mutations to inserting selectable markers. While
disrupting the function of a gene, researchers can elucidate the impact of the HDR technique requires further refinement, researchers have already
the knockout on cellular structure and function. In addition to knocking employed the method to correct a genetically-encoded mutation causing
out gene function, CRISPR can be used to delete large DNA fragments cataracts in mice (2), demonstrating proof of concept for HDR as a method
from the genome. These genomic deletions can be achieved by using two for correcting genetically-based diseases.
gRNAs that direct Cas9s to simultaneously create DSBs at opposite ends of
the DNA fragment to be excised. Scientists at Caribou Biosciences recently CRISPR interference (CRISPRi)
discovered that the size and nature of the errors made during NHEJ are in
fact not random, but depend on the target sequence (1). This knowledge While genetic knockouts can be achieved by disrupting a genetic sequence,
could allow us to further exploit the error-prone repair machinery to predict gene expression can be altered without altering the corresponding DNA.
repair outcomes and edit DNA precisely. By mutating the endonuclease domain of Cas9, the enzyme can be made
catalytically inactive (i.e., dead Cas9 or dCas9). Researchers have used
To learn about how to experimentally make knockout cells, download our dCas9 to develop a technique wherein the CRISPR complex binds to its DNA
Cell Engineering 101 eBook. target but does not cleave it. The binding of the dCas9 interferes with gene
expression by preventing the cell’s transcription machinery from accessing

Knock-ins the gene, thereby silencing its expression. Fusing a transcriptional repressor
protein to dCas9 allows reversible and fine-tuned reduction in gene
The incorporation of genetic material into a cell’s genome is referred to as expression.

Knockout (KO): a mutation in a genetic sequence that Knock-in (KI): the integration of a foreign genetic CRISPRi: a gene-silencing technique whereby a gRNA
causes it to be inoperative (i.e., no functional protein is sequence into a cell’s genome. binds to a target and an associated dead Cas9 blocks
made). transcription machinery from accessing the DNA..

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In a nod to the precursor gene-silencing technique, RNA interference Neisseria meningitidis (Nme). In fact, the team showed inhibition of the
(RNAi), the CRISPR silencing technique has been termed CRISPR Nme-Cas9 activity and binding in bacterial and mammalian cells using
interference, or CRISPRi (3). Compared to RNAi, CRISPRi is associated these anti-CRISPR proteins (6). Ultimately, this technology can be used to
with higher efficiency, greater versatility, and fewer off-target effects. reduce editing errors. It turns out that adding anti-CRISPR proteins after
editing takes place only partially reduces cleavage at on-target sites, but

CRISPR activation (CRISPRa) greatly reduces cleavage at off-target sites.

While dCas9 endonucleases can be used to silence gene expression, CRISPR Screens
they can also be further modified to enable fine-tuned control over
the activation of target genes (called CRISPR activation or CRISPRa). By Another application of CRISPR technology is in genome-wide functional
fusing a transcriptional activator to dCas9, scientists are now developing screening. Until recently, RNAi was the primary approach for performing
systems that can overexpress genes of interest. Polstein and Gersbach such screens, whereby genes are systematically inhibited across the
created one such system by fusing light-inducible proteins to mutated genome in order to determine their associated function and phenotype.
Cas9, causing gene expression to be activated in the presence of blue However, as mentioned previously, RNAi is plagued with problems
light and repressed in its absence (4). Other teams of researchers, such related to low efficiency and high off-target effects. With the advent
as Zalatan et al., are building more complex systems for multiplexed of CRISPR, genomic screening libraries are now being developed and
gene activation and repression at as many as three loci simultaneously applied to knockout thousands of genes in a single screen with high
(5). efficiency. Schmidt and colleagues recently developed a CRISPR screening
library based on HEK293T cells that targets nearly every known protein-

Anti-CRISPR coding gene in the human genome and that they are making freely
available to the research community (7).
Although the CRISPR-Cas9 system enables fine-tuning of genomic DNA,
one downside is the risk of off-target effects (cutting DNA in the wrong Gene Visualization
place). One solution to this issue is to harness “anti-CRISPR” proteins that
inhibit Cas9 activity. In nature, phages use these proteins to evade the The CRISPR system can also be used to visualize genomic regions. This is
CRISPR machinery of the bacteria. Recently, Pawluk et al. discovered anti- achieved by attaching fluorescent proteins (like GFP) to dCas9 proteins
CRISPR protein inhibitors that were effective against Cas9 nuclease from and using the CRISPR system to tag desired parts of the genome. (8,9).

CRISPRa: a gene-activation technique whereby a transcriptional activator is complexed with anti -CRISPR: proteins that prevent Cas9 from cutting DNA. Anti-CRISPR can be used to
dCas9 to increase the expression of a gene of interest. reduce off-target effects while maintaining on-target activity.

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Multi-guide Multi-guide sgRNAs: multiple gRNAs designed to target the same gene and are introduced
to cells simultaneously.
Scientists can introduce multiple gRNAs that target the same gene (multi-
guide) into cells at once. This method has been shown to induce large
fragment deletions that can efficiently knock out genes. Alternatively,
gRNAs can be used to edit several genes simultaneously (10,11).

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The Full Stack Approach to Genome Engineering

A CRISPR experiment involves three basic steps:
Design guide RNAs, Edit DNA, and Analyze results.

The end-to-end solutions of this workflow represent a "full stack"

approach to genome engineering. The solutions for each step are
outlined below discussed in the following sections.

Design
The first step of a CRISPR experiment involves designing gRNAs and
choosing an appropriate Cas9 nuclease.

Edit
The gRNAs and Cas9 are delivered to cells so that genome editing can
take place. The format of the CRISPR components and method of delivery
into cells are important factors in this step.

Analyze
After CRISPR editing, the targeted genomic sequences must be analyzed
to assess the frequency and type of edits made. Obtaining high
frequencies of the desired edit is highly dependent on the design and
edit steps of the workflow.

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Design
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Choosing the Right CRISPR For knock-in, CRISPRa, and CRISPRi experiments, gRNAs have additional
requirements. For knock-in experiments, the template DNA should be
Components carefully checked for PAM sequences to ensure that it is not treated as
a target. For CRISPRa/ CRISPRi applications, the gRNA sequence should
The design of gRNA and choice of the nuclease be complementary to the promoter regulating the target gene, rather to
depends on the desired application. The right choice of sequence of the gene itself.
components is crucial for a successful experiment.
Design Tools
Designing the Guide RNA Several online tools are available to help design guide RNAs by predicting
their on-target and off-target activity. We have listed some of these
The guide RNA is the programmable component of the CRISPR-Cas9
below.
system that directs the Cas9 nuclease to the target site. Therefore, it is
important to ensure that the guide sequence yields minimum off-target
Synthego CRISPR Design Tool
(unintended) cuts while also providing maximum on-target activity.
Synthego’s free CRISPR design tool is one of the fastest and most efficient design tools
available for researchers. The tool offers easy design of synthetic sgRNAs with up to
Stringency of additional parameters for RNA sequence design further 97% editing efficiency and the lowest off-target effects. It includes a library of more
than 100,000 genomes and 9,000 species, and offers a convenient way to order your
varies with the intended applications. For instance, knock out procedures
guides within the tool. You can also use the tool to validate gRNAs designed using
rely on cells forming frameshifts in the DNA during repair of the DSB by other platforms.
NHEJ. The faulty reading frame disrupts transcription of the gene, and
thereby downstream protein coding. These experiments offer some MIT CRISPR Designer
flexibility in terms of sequence selection and target location of the guide The MIT CRISPR designer is another free platform that helps users design gRNAs with
high target selectivity and low off-target effects.
RNA. Nevertheless, the following criteria should be met when designing
guide RNAs for KO experiments:
sgRNA Scorer 2.0
The sgRNA scorer 2.0 from the Dr. George Church’s lab identifies target sites for gRNA
• Choose guide sequences that target exons that are present in all from an input DNA sequence. The tool currently allows selection from six different
nucleases, and predicts the activity of Cas9 nucleases derived from S. aureus and S.
transcript variants. thermophilus 3 especially well.

• Choose sequences at the 5’ end of the gene or that encode essential Benchling
protein domains, as these are more likely to interfere with protein Benchling’s cutting edge design tool conveniently integrates other tools so that
scientists can use a single interface for designing their experiment. While the basic
function. plan is free, users need to pay fees for accessing their Startup and Enterprise plans.

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Selecting a Nuclease
Several types of nucleases are now available for CRISPR applications. Selecting one depends on the specific goals and
limitations of the experiment. The four nucleases described below facilitate many types of genome editing projects. As
additional nucleases are discovered, we can expect CRISPR editing to become even more flexible and more powerful
than it is today.

S. pyogenes Cas9 (SpCas9) Cpf1

The Cas9 nuclease from the bacteria Streptococcus pyogenes (SpCas9) Another common nuclease is Cpf1, short for ‘CRISPR from Prevotella
is the most commonly used nuclease in CRISPR genome engineering and Francisella after the bacterial species from which it originates. Cpf1
assays. This nuclease recognizes the PAM motif 5’-NGG-3’ (N signifies any functionally differs from SpCas9 in three key aspects: 1) Cpf1 recognizes
nucleotide) and creates a DSB with blunt ends at the target site. It was and binds to the PAM motif, 5’-TTN-3’. It therefore can be a better choice
the first nuclease engineered for CRISPR editing and is used for a variety for targeting DNA regions with high AT-content than Cas9. 2) Cpf1 creates
of knockout and knock-in applications. a staggered double-stranded (overhangs), rather than the blunt-end
cut generated by its SpCas9 counterpart. Thus Cpf1 is preferred for
Cas9 Nickase experiments relying on the HDR repair outcome. 3) Cpf1 is a smaller
protein than SpCas9 and does not require a tracrRNA. Thus, the gRNA
Cas9 nickase is a modified version of the Cas9 protein. Instead of creating
required by Cpf1 is shorter in length and cheaper to generate than the
a DSB, this nuclease nicks a single DNA strand. Because a nick in each
gRNA required by SpCas9.
DNA strand is needed to create a DSB, there are typically fewer off-target
effects relative to using an unmodified Cas9 nuclease. Additionally, this
nuclease makes a staggered cut that leaves long overhangs instead of S. aureus Cas9 (SaCas9)
blunt ends at the cut site. This enables more control when inserting a Cas9 from Staphylococcus aureus (SaCas9) is an ortholog of the Cas9
DNA segment for HDR repair. For this reason, Cas9 nickase is often used family. SaCas9 recognizes the PAM sequence of 5’-NNGRRT-3’. However,
in knock-in experiments. the SaCas9 protein is much smaller than SpCas9, the sequences that
encode them differ by length of about 1kb. Its small size allows SaCas9 to
be packaged into an AAV vector for cellular delivery, an approach that is
difficult with the large SpCas9.

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Edit
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of Cas9 within the cells. Plasmids also run the risk of integrating into
Different Formats of CRISPR Guides
A CRISPR genome editing experiment can be
genome of the host cell in places that cause cytotoxicity. This is
orchestrated in several different ways. The essential
particularly problematic in applications related to human medicine and
CRISPR machinery (gRNA and Cas9 protein) can be crop engineering.
delivered in one of the three formats: plasmid DNA,
in vitro-transcribed RNA, or ribonucleoprotein (RNP) RNA
complex. The CRISPR components can be delivered into the cells in RNA format:
gRNA and RNA encoding the Cas9 protein. The RNA can be produced
DNA enzymatically through in vitro transcription (IVT) or through a synthetic
One approach is to deliver CRISPR components into the cells as plasmid process.
DNA. This approach requires a researcher to clone both the Cas9 protein
and desired gRNA DNA fragments into a plasmid, and then introducing In vitro transcription (IVT)
the plasmid into target cells. Commercial plug-and-play plasmids are The RNA can be enzymatically transcribed from the corresponding DNA
now available for this purpose, allowing researchers to insert a gRNA outside the cell in a process called in vitro transcription (IVT). The first
sequence with their design specifications. step is same as that in plasmid cloning: design the DNA template based
on the target sequence within the gene of interest. In this case, the
The first step involves designing the DNA template to encode the gRNA template must additionally include an upstream promoter site (typically
and Cas9 protein. Once the DNA template sequence has been designed, T7) for the RNA polymerase to use during transcription.
an oligo can be ordered and cloned into a plasmid. Engineering a CRISPR
plasmid follows the same protocol as a typical cloning assay. The DNA Once the DNA template has been obtained, it is then transcribed into a
template insert must be amplified, digested, and ligated with the plasmid gRNA using one of multiple IVT kits available on the market (containing
before being transformed into cells. After screening for the recombinant enzymes and reagents). The IVT process requires 1-3 days to prepare
plasmid and verifying its sequence, the plasmid can be delivered to the the gRNA for a CRISPR assay. After purification, the gRNA can be co-
target cell. transfected into the cell alongside Cas9 mRNA. Alternatively, it can
also be complexed with Cas9 protein and delivered to the target cell as
Overall, the process of preparing a custom CRISPR plasmid in-house ribonucleoproteins (RNPs).
consumes 1–2 weeks of time before the actual editing assay can be
undertaken. In addition to the excessive time investment, the plasmid The IVT approach is laborious and not scalable for multiple CRISPR
approach is also prone to off-target effects due to the continual presence targets. In addition, IVT-derived gRNAs are prone to mistakes made by

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enzymes during synthesis. For instance, RNA polymerase may insert the be introduced with Cas9 protein as ribonucleoprotein complexes, as
wrong nucleotide, or make a guide sequence too long or short. This may described below.
not only lead to highly variable editing efficiencies but also increase the
possibility of off-target effects. Ribonucleoprotein (RNP)
Deploying gRNA with Cas9 protein as ribonucleoprotein (RNP) complexes
Synthetic guide RNA
is the most effective strategy, as researchers have demonstrated higher
A convenient substitute to the IVT-derived guide RNA involves synthetic editing efficiencies and fewer potential off-target effects using RNPs as
polymerization of high-quality RNA (up to 120-mer) sequences. These compared to other delivery methods (11). Moreover, as the RNP exists
can either be in the form of separate crRNA and tracrRNA fragments that transiently inside the cell (not inserted into the genome), there is low risk
must be annealed together, or as seamless single guide RNA (sgRNA). Of of cytotoxicity.
these two forms, sgRNA generally produces a higher editing efficiency
by avoiding the inefficient annealing of the two-piece system, and the It has become clear that the RNP format is the most effective option for
tendency of tracrRNA fragments to form tetramers that interfere with the carrying out CRISPR gene editing assays and other applications, especially
Cas9 protein. in embryos.

Until very recently, the cost associated with producing synthetic sgRNA
was high enough to make synthetic sgRNA a prohibitive expense for
many laboratories. However, recent developments in technology enable
affordable access and successful deployment of the CRISPR technology.
One such advancement lies in Synthego’s automated and scalable
production methods, which allows these sgRNAs to be generated
much more rapidly and at a much lower cost than traditional synthesis
methods. The high-throughput synthesis technique also results in higher
fidelity sgRNAs, as compared to IVT-derived guides, enabling more
Ribonucleoprotein (RNP): a complex guide RNA and Cas9 protein. A popular
efficient and reproducible targeting of the desired genomic site. transfection format for CRISPR editing.

In addition, Synthego’s synthesis platform enables production of

chemically modified sgRNAs. Such modifications protect the sgRNAs
from degradation by exonucleases and intracellular immune responses Chemically modified sgRNAs: synthetic single guide RNAs that have been
chemically modified to resist degradation within cells such that more editing can
within the cell, and thus enables more potential editing to take place. take place.
Synthetic sgRNA may be co-transfected with Cas9 mRNA or they may

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Table 2. Comparison of cost, time, and labor associated with different guide RNA formats

CRISPRevolution Modified sgRNA in vitro-transcribed (IVT) sgRNA Plasmid DNA

FL E X I B I L I T Y
stem cells primary cells cell lines stem cells primary cells cell lines stem cells primary cells cell lines

PR E P A R A T I ON S TEP S 3 5 5

PR E P A R A T I ON T I ME Low Medium High

NUM B E R O F C L ONE S TO S OR T Few Many Many

ED IT I N G E F F I C I ENC Y Consistently High Variable Variable

TOT A L C O S T TO G ENER A TE A K O $ $$$ $$$

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Delivering CRISPR Components Electroporation and Nucleofection

Inside Cells Electroporation enables delivery of the CRISPR machinery in cell
types that are difficult to transfect using lipofection. The application
Several different transfection methods can be used of a controlled, short electric pulse to the cells forms pores in the cell
membrane, allowing entry of foreign material. Nucleofection is a variant
to deliver CRISPR components to cells (Table 3). The
of electroporation, in which the electric pulse is optimized such that the
method used largely depends on the cell type and
nuclear membrane of the cells also forms pores. The CRISPR components
format of the CRISPR components. are thus directly delivered to the nucleus.

Lipofection Microinjection
In a lipid-based delivery system, cationic lipid reagents facilitate delivery
Microinjection is commonly used to inject the Cas9 and gRNA into
of biomolecules into cells. This method is high-throughput, has low
oocytes or zygotes in order to generate genetically engineered
cytotoxicity, and is applicable in various cell types. Traditionally used for
organisms. This technique can also be used to transfect some types of
delivering nucleic acids in cells, lipofection has recently been optimized
cells. Zebrafish, mouse, and human embryos have been manipulated
for delivering RNPs into cells.
using this technique.

Table 3. Comparison of CRISPR transfection methods

Lipofection Electroporation Nucleofection Microinjection Virus

Lipid complexed with genetic Electric pulse forms pores in An electroporation-based Microneedle injects CRISPR DNA/RNA is packaged into
material fuses with the cell the cell membrane for entry method that delivers materials components inside cells, infectious particles and
PR IN C I P L E
membrane. of DNA/RNA/RNP. directly to the nucleus pre- oocytes, or zygotes. introduced into cells.
optimized for each cell type.

• Cost effective • Easy, fast • Easy, fast • High efficiency • High efficiency
ADV A NTA G E S
• High throughput • High efficiency • High efficiency

• Less efficient • Requires optimization • Requires specialized • Time-consuming • Time-consuming

LI M I T A T I ONS • Requires specialized equipment • Technically demanding • Safety requirements
equipment • Low throughput • Expensive

CE LL T Y P E S Few Numerous Numerous Few Numerous

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Virus
Viral vectors can be used to transfer DNA or RNA into cells in a process
called transduction. There are several types of viruses that can be used
for transduction, including lentivirus, adenovirus, adeno-associated virus
(AAV), and herpes viruses. For stable transduction, lentiviruses, are often
used because they integrate their genome into the genome of infected
cells.

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Analyze
C RI SP R 1 0 1 - C R I S P R A n alysis

Analysis of CRISPR Editing

After transfecting the cells, the efficiency of DNA editing
using CRISPR needs to be determined.

The easiest way to do this is to Sanger-sequence the edited and control

DNA at the cut site and a software analysis program, such as Synthego’s
Inference of CRISPR Edits (ICE) tool or Tracking Indels by DEcomposition
(TIDE). Synthego’s ICE tool is a free and easy-to-use program that will give
results within minutes. From the ICE report, one can find out the overall
editing efficiency, the specific indels generated, and the Knockout Score
(percentage of indels will likely cause a knockout).

Although purely qualitative and somewhat outdated, DNA mismatch

detection assays are also an option. This approach involves the treatment
of the CRISPR-edited DNA and the non-CRISPR edited DNA (control) with
an enzyme that cuts DNA at mismatched sites. Gene deletions by the
CRISPR system often result in mismatched bases during DNA repair.
Thus, the CRISPR-edited DNA shows multiple small fragments after size-
based separation in a gel, while the control shows a single band of uncut
DNA. This method is a simple and crude way of estimating the CRISPR
editing efficiency.

Lastly, next generation sequencing (NGS) is a gene sequencing method

Inference of CRISPR Edits (ICE): a free online tool that analyzes the editing and
that can be used for accurate and quantitative analysis of the editing knockout efficiencies of CRISPR experiments.
efficiency of CRISPR. It also provides additional information regarding off-
target edits in the DNA. However, NGS is an expensive option, especially
when Synthego’s ICE tool gives NGS-quality results for free.
Knockout Score: A metric of knockout efficiency determined by Synthego ICE
analysis. KO Score is defined as the percentage of sequences in a CRISPR edited
pool that lead to a putative knockout, including frameshift-inducing indels and
indels that are 21+nt in length or larger.

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CRISPR in the Future

CRISPR has received a lot of attention primarily due to its ability to genetically
edit living organisms. However, while this side of CRISPR occupies the
spotlight, researchers have begun tinkering with the technology to unlock its
vast potentials that go beyond the applications discussed so far.

Scientists are now using a modified version of CRISPR to explore epigenomics—the genome-wide
set of chemical groups that adorn DNA and its associated histone packaging proteins. Previously,
researchers were merely able to catalogue the correlation between epigenetic markers and gene
expression in cells. Now, a CRISPR complex that is capable of acetylating histone proteins at precise
locations dictated by the complex’s gRNA has been developed (12). Such technologies can shed light
on the causal relationship between epigenetic markers and gene expression in the future.

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CRISPR is also enabling the elucidation of large portions of the human genome, the function of Additional Information
the vast majority of which is unknown. Scientists have long been trying to identify the location For an up-to-date list of all Synthego Application
Notes and other resources, please visit
and function of ‘non-gene’ genetic elements that do not code for proteins but are thought to
Synthego.com/resources
have important regulatory roles in expression. CRISPR is allowing researchers to knock out these
For technical assistance,
previously uncharted regions to study their role in the cell (13). contact our Scientific Support Team

CRISPR is also playing an increasingly important role in the biomedical industry. The process of drug Ph: 888.611.6883

discovery is notoriously long, arduous, and expensive. CRISPR, however, is likely to expedite the Email: [email protected]
pre-clinical stage of developing new drugs. For instance, CRISPR screening libraries are now available
to facilitate the discovery new drug targets. CRISPR can also be used to develop accurate disease About Synthego
models to validate potential drugs. In parallel, research using CRISPR for in vivo and ex vivo therapies
Synthego is the leading genome engineering
is on the rise. There is a lot of excitement and optimism that that CRISPR will enable the development innovation company. The company’s automated,
of better therapies and more personalized medicine in the near future. full stack genome engineering platform enables
broader access to CRISPR to accelerate basic
scientific discovery, uncover cures for diseases,
CRISPR is not only paving the way for researchers to solve the most difficult of problems in the life and develop novel synthetic biology applications.
sciences, but it is also enabling the scientific community to explore dimensions of the genome that Headquartered in Silicon Valley, Synthego is used
by scientists from the largest global biotechnology
we’ve been unable to study up until this point. Due to its adaptability across a wide range of species
companies and global biology universities to unlock
and its simplicity of use, CRISPR-Cas9 has quickly revolutionized genome engineering. CRISPR-Cas9 the potential of gene editing.
technology promises to deliver some truly stunning advances within the coming decades, particularly
in relation to human therapeutics, agricultural biology, biofuels, and basic scientific research.

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References
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C, Reece-Hoyes JS, Nye C, Gradia S, Vidal B, Zheng J, Hoffman GR, Fuller library targeting the human genome. Sci Rep. 2015 Oct 8;5:14987. doi:
CK, May AP. DNA Repair Profiling Reveals Nonrandom Outcomes at 10.1038/srep14987. PubMed PMID: 26446710; PubMed Central PMCID:
Cas9-Mediated Breaks. Mol Cell. 2016 Aug 18;63(4):633-46. doi: 10.1016/j. PMC4597219.
molcel.2016.06.037. PubMed PMID: 27499295..
8. Ma, Hanhui, et al. Multicolor CRISPR labeling of chromosomal loci in
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24315440. 9. Ma, Hanhui, et al. Multiplexed labeling of genomic loci with dCas9 and
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PMID: 24136345; PubMed Central PMCID: PMC3922765. systems. Science 339.6121 (2013): 819-823.

4. Polstein LR, Gersbach CA. A light-inducible CRISPR-Cas9 system 11. Liang, Xiquan, et al. Rapid and highly efficient mammalian cell
for control of endogenous gene activation. Nat Chem Biol. 2015 engineering via Cas9 protein transfection. Journal of biotechnology 208
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12. Hilton IB, D’Ippolito AM, Vockley CM, Thakore PI, Crawford GE,
5. Zalatan JG, Lee ME, Almeida R, Gilbert LA, Whitehead EH, La Russa M, Reddy TE, Gersbach CA. Epigenome editing by a CRISPR-Cas9-based
Tsai JC, Weissman JS, Dueber JE, Qi LS, Lim WA. Engineering complex acetyltransferase activates genes from promoters and enhancers. Nat
synthetic transcriptional programs with CRISPR RNA scaffolds. Cell. Biotechnol. 2015 May;33(5):510-7. doi: 10.1038/nbt.3199. PubMed PMID:
2015 Jan 15;160(1-2):339-50. doi: 10.1016/j.cell.2014.11.052. PubMed PMID: 25849900; PubMed Central PMCID: PMC4430400.
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13. Ho TT, Zhou N, Huang J, Koirala P, Xu M, Fung R, Wu F, Mo YY. Targeting
6. Pawluk, April, et al. Naturally occurring off-switches for CRISPR-Cas9. Cell non-coding RNAs with the CRISPR/Cas9 system in human cell lines.
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