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CRISPR header icon CRISPR in the Lab: A Practical Guide


The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Type II system is currently the most commonly used RNA-Guided Endonuclease technology for genome engineering (see CRISPR history). There are two distinct components to this system: (1) a guide RNA and (2) an endonuclease, in this case the CRISPR associated (Cas) nuclease, Cas9. The guide RNA is a combination of the endogenous bacterial crRNA and tracrRNA into a single chimeric guide RNA (gRNA) transcript. The gRNA combines the targeting specificity of the crRNA with the scaffolding properties of the tracrRNA into a single transcript. When the gRNA and the Cas9 are expressed in the cell, the genomic target sequence can be modified or permanently disrupted (learn more about gRNA and Cas9 expression).

The gRNA/Cas9 complex is recruited to the target sequence by the base-pairing between the gRNA sequence and the complement to the target sequence in the genomic DNA. For successful binding of Cas9, the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence (learn more about PAM sequences). The binding of the gRNA/Cas9 complex localizes the Cas9 to the genomic target sequence so that the wild-type Cas9 can cut both strands of DNA causing a Double Strand Break (DSB). Cas9 will cut 3-4 nucleotides upstream of the PAM sequence. A DSB can be repaired through one of two general repair pathways: (1) the Non-Homologous End Joining (NHEJ) DNA repair pathway or (2) the Homology Directed Repair (HDR) pathway. The NHEJ repair pathway often results in inserts/deletions (InDels) at the DSB site that can lead to frameshifts and/or premature stop codons, effectively disrupting the open reading frame (ORF) of the targeted gene (Learn more about using CRISPR technology to disrupt a gene). The HDR pathway requires the presence of a repair template, which is used to fix the DSB. HDR faithfully copies the sequence of the repair template to the cut target sequence. Specific nucleotide changes can be introduced into a targeted gene by the use of HDR with a repair template (Learn more about using CRISPR technology to edit a gene).


Disrupt Your Gene of Interest (via Insertions / Deletions)

The Cas9 nuclease has two functional domains: RuvC and HNH, each cutting a different DNA strand. When both of these domains are active, the Cas9 causes double strand breaks (DSBs) in the genomic DNA. In the absence of a suitable repair template, the DSB is repaired by the Non-Homologous End Joining (NHEJ) DNA repair pathway. During NHEJ repair, InDels (insertions/deletions) may occur as a small number of nucleotides are either inserted or deleted at random at the DSB site. InDels alter the Open Reading Frame (ORF) of the target gene, which may significantly change the amino acid sequence downstream of the DSB. Additionally, InDels could also introduce a premature stop codon either by creating one at the DSB or by shifting the reading frame to create one downstream of the DSB. Any of these outcomes of the NHEJ repair pathway can be leveraged by scientists to disrupt their target gene. It is important to note that the InDels induced by NHEJ will be random, so the type and extent of gene disruption will need to be determined experimentally.


In order to maximize the effect of gene disruption, target sequences should be chosen near the N-terminus of the coding region of the gene of interest (Learn more about Designing gRNAs). Typically, the target sequence is selected to introduce a DSB within the first or second exon of the gene. It is important not to design targets to introns (non-coding regions), as repair of the DSB in that region will not disrupt the target gene. The changes introduced by this use of the CRISPR system are permanent to the genomic DNA of the organism.

Plasmids: Double-strand Break (Cut)

Concerned about Off-Target Effects? Browse our CRISPR Nickase plasmids for single-strand breaks: Single-strand Break (Nick)

Edit / Modify the Endogenous Genome (via Homology Directed Repair)

The CRISPR system can also be used to introduce specific nucleotide modifications at the target sequence. While NHEJ repair is imperfect and often results in a disruption of the Open Reading Frame of the gene, cells can utilize a less error-prone DNA repair mechanism: Homology Directed Repair (HDR). Read more about specific uses of HDR: Mol Cell Biol. Jan 1998 and G3 (Bethesda). Apr 2013

In order to introduce nucleotide modifications to genomic DNA, a DNA repair template containing the desired sequence must be present during HDR. The DNA template is normally transfected into the cell along with the gRNA/Cas9 and must have a high degree of homology to the sequence immediately upstream and downstream of the DSB. The length and binding position of each homology arm is dependent on the size of the change being introduced (See CRISPR FAQs for details). In the presence of a suitable template, HDR can faithfully introduce specific nucleotide changes at the Cas9 induced DSB. The desired modification in the genomic DNA must be confirmed experimentally.


When designing a repair template for genome editing by HDR, it is important that the repair template does NOT contain the target sequence followed by the PAM sequence or the template itself will also be cut by the Cas9. Changing the sequence of the PAM in the repair template should be sufficient to ensure it is not cut by Cas9.

Plasmids: Double-strand Break (Cut)

Concerned about Off-Target Effects? Browse our CRISPR Nickase plasmids for single-strand breaks: Single-strand Break (Nick)

Off-Target Effects and Cas9 Nickase

The CRISPR technology is becoming widely-used because of its ease of use and efficacy. However, off-target effects of the Cas9 nuclease activity is a current concern with the use of the CRISPR system. Apparent flexibility in the the base-pairing interactions between the gRNA sequence and the genomic DNA target sequence allows imperfect matches to the target sequence to be cut by Cas9. Single mismatches at the 5’ end of the gRNA (furthest from the PAM site) can be permissive for off-target cleavage by Cas9.

Modified versions of the Cas9 enzyme containing a single inactive catalytic domain, either RuvC- or HNH-, are called ‘nickases’. With only one active nuclease domain, the Cas9 nickase cuts only one strand of the target DNA, creating a single-strand break or 'nick'. Similar to the inactive dCas9 (RuvC- and HNH-), a Cas9 nickase is still able to bind DNA based on gRNA specificity, though nickases will only cut one of the DNA strands. The majority of CRISPR plasmids currently being used are derived from S. pyogenes and the RuvC domain can be inactivated by a D10A mutation and the HNH domain can be inactivated by an H840A mutation.

A single-strand break, or nick, is normally quickly repaired through the HDR pathway, using the intact complementary DNA strand as the template. However, two proximal, opposite strand nicks introduced by a Cas9 nickase are treated as a Double Strand Break (DSB), in what is often referred to as a 'double nick' or 'dual nickase' CRISPR system. A double-nick induced DSB can be repaired by either NHEJ or HDR depending on the desired effect on the gene target. See the sections on Gene Editing (HDR) or Gene Disruption (InDel) for more information.


In the figure, two different gRNAs (#1 and #2) bind in a particular genomic region. When gRNA #1 and #2 are co-expressed with a Cas9 nickase, single-strand nicks are created in the DNA at (A), (B) and (C). The nick created at (A) is quickly repaired by HDR using the intact compliment strand as a template and no change occurs. The nicks at (B) and (C) are in close proximity (and on opposite strands) and together behave as a DSB.

If specificity and reduced off-target effects are crucial, consider using the Cas9 nickase to create a double nick-induced DSB. By designing two gRNAs with target sequences in close proximity and on opposite strands of the genomic DNA, off-target effects of either gRNA alone will result in nicks that will not change the genomic DNA. Only at the target location where both nicks are proximal, will the double nicked sequence be considered a DSB. For more information on the double-nickase technique, read Ran et al. Cell. 2013 Sep 12. PubMed.

Cas9 Version Guide RNA requirement InDels induced? HDR induced? Recommended Application
Wildtype (Cut) Cas9 (WT) One gRNA for each target Yes, most efficient Yes Guide screening, InDel generation, HDR (when accuracy, i.e. off-target effects, is not a big concern)
Single Nickase Cas9 (D10A) One gRNA for each target Barely detectable Yes, less efficient HDR without InDel generation
Double Nickase Cas9 (D10A) Two gRNAs for each target Yes, very efficient Yes InDel generation, HDR (when accuracy, i.e. off-target effects, is a concern)

Plasmids: Double-strand Break (Cut), Single-strand Break (Nick)

Expanded Uses of the CRISPR System for Genome Manipulation

The CRISPR/Cas system is a remarkably flexible tool for genome manipulation. One of the primary advantages of the technology is that the nuclease activity and the DNA-binding activity of Cas9 are discrete functions in the protein. The Cas9 nuclease activity (cut) is performed by 2 separate domains, RuvC and HNH. Each domain cuts one strand of DNA and each can be inactivated by a single point mutation. In S. pyogenes, a Cas9 D10A mutant has an inactive RuvC domain (RuvC-) and an active HNH domain (HNH+) and a Cas9 H840A mutant has an inactive HNH domain (HNH-) and an active RuvC domain (RuvC+). When both domains are inactive (D10A and H840A, RuvC- and HNH-) the Cas9 has no nuclease activity (catalytically inactive) and is said to be 'dead' (dCas9); however, the inactive dCas9 still retains the ability to bind to DNA based on gRNA specificity.

Activate or Repress Gene Expression

The dCas9 can be utilized as a platform for DNA transcriptional regulators to activate or repress gene expression by fusing the inactive enzyme (that retains its gRNA-binding ability) to known regulatory domains. The transcriptional activator VP64, when fused to dCas9, is capable of up-regulating gene transcription of targeted genes to enhance expression. Conversely, the binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription. Fusions of dCas9 with transcriptional repressors, such as KRAB, have been developed to further improve the silencing capabilities of the CRISPR system. By choosing a gRNA binding site near the promoter region of a gene, researchers can artificially activate or repress the transcription of a gene.


Unlike the genome modifications induced by CRISPR Gene Editing or CRISPR Gene Disruption, CRISPR-induced gene activation or repression is not inherently permanent, since it does not directly affect the genomic DNA.

Plasmids: Activate , Repress / Interfere

Purify Regions of Genomic DNA

Building on the well-established concept of ChIP (Chromatin Immunoprecipitation), researchers have created enChIP (engineered DNA-binding molecule-mediated ChIP) that allows for the purification of any genomic sequence specified by a particular gRNA. A catalytically inactive dCas9 fused to an epitope tag(s) can be used to purify genomic DNA bound by the gRNA. Learn more about ChIP here.

Plasmids: Purify

Label Regions of Genomic DNA for Imaging

Using a dCas9 fused to a fluorescent marker (such as GFP), researchers have turned dCas9 into a customizable DNA label that can be detected in live cells. By creating unique gRNAs that bind in close proximity along a stretch of genomic DNA, a technique refered to as 'tiling', researchers have imaged specific regions of the genome. The tiling technique does require multiple gRNAs to bind near one another in order to produce a detectable signal.

Plasmids: Label

Genomic Screens with Pooled Libraries

Using the CRISPR system, researchers have created pooled libraries of gRNAs that can be used in powerful screening techniques. Learn More about pooled libraries and how to use them.

Plasmids: Screen using pooled gRNA Libraries

Designing gRNA Targets

As versatile as the Cas9 protein is (as either a nuclease, nickase or platform), it requires the targeting specificity of a gRNA in order to act. Choosing an appropriate target sequence in the genomic DNA is a very important step in designing your experiment. The target sequence is 20 nucleotides followed by the appropriate Protospacer Adjacent Motif (PAM) sequence in the genomic DNA. Target sequences (20 nucleotides + PAM) can be on either strand of the genomic DNA. Target sequences can appear in multiple places in the genome, which is why the use of a bioinformatic program to help choose target sequences and minimize off-target effects is highly recommended. There are a number of tools available to help choose/design target sequences as well as lists of bioinformatically determined (but not experimentally validated) unique gRNAs for different genes in different species. The following links are free resources:

Software for Designing gRNAs

  • Feng Zhang lab's Target Finder Identifies gRNA target sequences from an input sequence and checks for off-target binding. Currently supports: Drosophila, Arabidopsis, zebrafish, C. elegans, mouse, human, rat, rabbit, pig, possum, chicken, dog, mosquito, and stickleback.

  • Michael Boutros lab's Target Finder (E-CRISP) Identifies gRNA target sequences from an input sequence and checks for off-target binding. Currently supports: Drosophila, Arabidopsis, zebrafish, C. elegans , mouse, human, rat, yeast, frog, Brachypodium distachyon, Oryza sativa, Oryzias latipes

  • RGEN Tools: Cas-OFFinder Identifies gRNA target sequences from an input sequence and checks for off-target binding. Currently supports: Drosophila, Arabidopsis, zebrafish, C. elegans , mouse, human, rat, cow, dog, pig, Thale cress, rice (Oryza sativa), tomato, corn, monkey (macaca mulatta).

  • CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes Identifies gRNA target sequences from an input sequence, checks for off-target binding and can work for S. pyogenes, S. thermophilus or N. meningitidis Cas9 PAMs. Currently supports: mouse and human.

  • CRISPR Optimal Target Finder Identifies gRNA target sequences from an input sequence and checks for off-target binding. Currently supports over 20 model and non-model invertebrate species.

Helpful Tips

The Protospacer Adjacent Motif (PAM) Sequence

For Cas9 to successfully bind to DNA, the target sequence in the genomic DNA must be complementary to the gRNA sequence and must be immediately followed by the correct protospacer adjacent motif or PAM sequence. The PAM sequence is present in the DNA target sequence but not in the gRNA sequence. Any DNA sequence with the correct target sequence followed by the PAM sequence will be bound by Cas9.


In the figure, the target sequence is followed by the PAM sequence at two separate locations (B and E). Cas9 will ONLY cut at B and E. The presence of the target sequence without the PAM following it (C and D) is NOT sufficient for Cas9 to cut. The presence of the PAM sequence alone (A) is not sufficient for Cas9 to cut.

The PAM sequence varies by the species of the bacteria from which the Cas9 was derived. The most widely used Type II CRISPR system is derived from S. pyogenes and the PAM sequence is NGG located on the immediate 3’ end of the gRNA recognition sequence. The PAM sequences of other Type II CRISPR systems from different bacterial species are listed in the Table below. It is important to note that the components (gRNA, Cas9) derived from different bacteria will not function together. Example: S. pyogenes (SP) derived gRNA will not function with a N. meningitidis (NM) derived Cas9.

Ran et al recently described a Cas9 from Staphylococcus aureus(SaCas9) that has similar genome editing efficiency to that of SpCas9, but is 1kb smaller. The smaller size of SaCas9 is ideal for adeno-associated virus (AAV) delivery, which has a packaging limitation of ~4.5kb.

Species PAM Sequence
Streptococcus pyogenes (SP) NGG
Staphylococcus aureus (SA) NNGRRT [also: NNGRR(N) should be considered]
Neisseria meningitidis (NM) NNNNGATT
Streptococcus thermophilus (ST) NNAGAAW
Treponema denticola (TD) NAAAAC

The majority of the CRISPR plasmids in Addgene’s collection are from S. pyogenes unless otherwise noted.

gRNA and Cas9 Expression

To use the CRISPR system, you will need both gRNA and Cas9 expressed in your target cells. Some research groups have experimented with codon-optimized versions of Cas9, in an attempt to increase efficiency of Cas9 expression. To date, codon-optimization of Cas9 appears to have a negligible effect on expression. The respective promoters for Cas9 and gRNA expression will ultimately determine the species specificity of a particular system. The figure below summarizes the product from each expression cassette. Addgene's CRISPR plasmids can either contain both cassettes on a single plasmid or the cassettes are expressed from two separate plasmids.


Many of the labs that have deposited plasmids to Addgene have provided protocols for cloning custom target sequences into their empty gRNA expression plasmids. Please note that protocols are specific to certain plasmids and are not interchangeable among empty gRNA expression plasmids.


CRISPR Associated Protein, the Cas9 nuclease is the active enzyme for the Type II CRISPR system.
Clustered Regularly Interspaced Short Palendromic Repeat, a region in bacterial genomes used in pathogen defense.
CRISPR Interference, using a dCas9+gRNA to repress/decrease transcription of a gene by blocking RNA Pol II binding.
the endogenous bacterial RNA that confers target specificity, requires tracrRNA to bind to Cas9.
a double strand break, the wild type function of Cas9.
dead Cas9, an inactive form of Cas9 that cannot cleave DNA.
Double Strand Break, a break in both strands of DNA, Cut, 2 proximal, opposite strand nicks can be treated like a DSB.
Dual Nick(ase)/Double Nick/Double Nicking:
a method to decrease off-target effects by using a single Cas9 nickase and 2 different gRNAs, which bind in close proximity on opposite strands of the DNA, to create a DSB.
engineered DNA-binding molecule-mediated ChIP, using a tagged-dCas9+gRNA to purify specific genomic regions to identify molecules associated with the genomic regions.
guide RNA, a fusion of the crRNA and tracrRNA, provides both targeting specificity and scaffolding/binding ability for Cas9 nuclease, does not exist in nature.
gRNA sequence:
the 20 nucleotides that precede the PAM sequence in the genomic DNA, what gets put into a gRNA expression plasmid, does NOT include the PAM sequence.
Homology Directed Repair, a DNA repair mechanism that uses a template to repair nicks or DSBs.
Insertion/Deletion, a type of mutation that can result in the disruption of a gene by shifting the ORF and/or creating premature stop codons.
Non-Homologous End-Joining, a DNA repair mechanism that often introduces InDels.
a break in only one strand of a double stranded DNA, normally repaired by HDR.
Cas9 that has one of the two nuclease domains inactivated. Can be either the RuvC or HNH domain.
Off-target effects:
gRNA binding to target sequences that do not match exactly, causing Cas9 to function in an unintended location, can be minimized by double-nick.
Open Reading Frame, the codons that make up a gene.
Protospacer Adjacent Motif, required sequence that must immediately follow the gRNA recognition sequence but is NOT in the gRNA.
RNA Guided EndoNuclease, the use of Cas9 and a gRNA, CRISPR technology.
single guide RNA, the same as a gRNA, which is a single stranded RNA.
Target sequence:
the 20 nucleotides that are incorporated into the gRNA plus the PAM sequence, target sequence is in the genomic DNA.
the endogenous bacterial RNA that links the crRNA to the Cas9 nuclease, can bind any crRNA.