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CRISPR Cas9 - gRNA Design

35 min Read
Video Summary

The latest tool in genome editing is the RNA-guided CRISPR Cas9 system which allows for highly specific genomic modifications. This groundbreaking technology has revolutionized genetic engineering through its ease of use, allowing targeting of any genomic loci simply by designing a 20 base pair RNA oligonucleotide. Guide RNA (gRNA) or short guide RNA (sgRNA) is an important component of the CRISPR Cas9 system and many considerations need to be taken when designing the gRNA sequence.

crRNA, tracrRNA, and gRNA

In bacteria and archaea, the CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA) form a complex which acts as the homing device for directing the Cas9 nuclease to invading foreign genetic materials (for more information visit CRISPR Cas9 – Introduction). By combining tracrRNA and crRNA into a single synthetic gRNA, this resulting one-component system not only simplifies the experimental design, it also yields equal or greater guiding efficiency (Figure 1) (1). Different constructs of gRNA have been designed and tested, each with different lengths of tracrRNA sequence at their 3’ end. The editing rate increases as the portion of the original tracrRNA sequence increases in the overall gRNA design (2). The most commonly used gRNA is about 100 base pairs in length. By altering the 20 base pairs towards the 5’ end of the gRNA, the CRISPR Cas9 system can be targeted towards any genomic region complementary to that sequence.

An example of the crRNA-tracrRNA complex and a gRNA

Figure 1 – An example of the crRNA-tracrRNA complex and a gRNA.

Our CRISPR-sgRNA vector design maximizes the editing efficiency in our lentiviral, adenoviral, and AAV formats.
General Guidelines for gRNA Design

The targeting specificity of the CRISPR Cas9 system is determined by the 20 nucleotide sequence at the 5’ end of the gRNA. For the S. pyogenes CRISPR Cas9 system, the desired target sequence must immediately precede a 5’-NGG protospacer adjacent motif (PAM - see CRISPR Cas9 - Introduction for more information). The gRNA guides the Cas9 nuclease to the target sequence by complementary base pairing and Cas9 nuclease mediates a double strand break ~3 nucleotides upstream of the PAM sequence (Figure 2). Note that the PAM sequence is not a part of the 20 base pair gRNA sequence, however, its presence in the genomic DNA is essential for CRISPR Cas9 genome editing.

sgRNA in the spCas9 system

Figure 2 – For the S. pyogenes system, the target sequence must be immediately adjacent to a 5’-NGG PAM sequence. The gRNA will form complementary base paring with the opposite strand of the target sequence and mediate a double strand break ~3bp upstream of the PAM sequence through the Cas9 nuclease.

As some promoters can restrict target site selection and gRNA design, it is imperative to choose a suitable promoter to drive the sgRNA expression. For example, the RNA polymerase III-dependent U6 promoter or the T7 promoter require a G or GG, respectively, at the 5’ end of the RNA sequence to initiate transcription (2). Therefore, if the U6 or T7 promoter is used in a S. pyogenes CRISPR system, then the target sequence is limited to the following forms respectively: GN16-19NGG or GGN15-18NGG. However, it is possible to bypass this restriction by simply adding the extra G or GG to the 5’ end of the 20 base pair guiding sequence (Figure 3).

CRISPR Cas9 - sequence restrictions imposed by use of the U6 or T7 Promoter

Figure 3 – Sequence restrictions imposed by the use of U6 or T7 RNA polymerase III promoters for gRNA expression can be by passed as illustrated here.

When designing the 20 base pair guiding sequence of the gRNA, the following three points should be taken into consideration: 1) GC content: the typical range is between 40% - 80% GC content where a higher GC content stabilizes the RNA:DNA duplex while destabilizing off-target hybridization; 2) length: the length could be adjusted and range from 17-24 base pairs, with shorter sequences leading to minimized off-target effects (3); and 3) potential off-target sites of the designed gRNA.

Mismatch tolerance between the gRNA and target site can lead to off-target effects of the CRISPR Cas9 system and in general, their occurrence depends on the following:

  • Mismatches at the 5’ end of the guiding sequence are more tolerated than in the first 8-14 base pairs.
  • More than 3 mismatches between the gRNA and target sequence will inhibit off-target effects.
  • Off-target effects also depend on the sequence of the gRNA with some gRNAs exhibiting less tolerance to mismatches than others.
  • The amount and the ratio between the Cas9 and gRNA introduced to a cell also impact off-target effects and careful titration of the Cas9 and gRNA can reduce these effects (4).

For the purpose of creating a small InDel through non-homologous end joining (NHEJ), there are many possible target sites across any protein. Targeting closer to the N’ terminus of a protein coding region rather than the C’ terminus is more desirable, because a frameshift is more likely to be deleterious if most of the protein has not yet been translated. Also, both the coding and non-coding strand of the genomic DNA can be targeted as they will both be equally efficient at creating InDel mutations. If homology directed repair (HDR) is needed for genome editing, however, the choice of target site is far more constrained by the desired location of insertion.

The various requirements outlined above need to be considered carefully when designing gRNAs. Carefully designed gRNA will lead to higher editing efficiency while minimizing off-target mutagenesis. Our Tools for gRNA Design article outlines tools available to any researchers to simplify the process of finding the perfect gRNA for any application.

Looking to design sgRNA for your gene? Check out our ready-to-use sgRNA lentivectors provided in a set of 3 here.
Special Design Considerations

Further considerations need to be taken when using paired Cas9 nickase induced double strand break. When designing gRNAs for paired nickase activity, it is important to note the following (Figure 4):

  • Ideally 5’ overhangs need to be generated upon nicking.
  • The space between the two gRNA can vary from being directly adjacent to each other or as far as 20 base pairs. The space should not exceed 20 base pairs.
  • The two gRNAs need to be designed based on target sequences on opposite strand. Note that the PAM sequence needs to be immediately upstream from the target sequence (5).

After the two gRNAs have been designed they could be mixed at a 1:1 ratio and delivered into the desired cell along with the Cas9 Nickase (D10A mutated Cas9) – see CRISPR Cas9 – Methods and Tools article for a detailed description of available delivery methods.

CRISPR Cas9 - Paired Nickase

Figure 4 – When using paired Cas9 nickases additional considerations need to be given to the gRNA design. These include producing a 5’ overhang, the spacing between the two gRNAs, and the relative position of the two gRNA target sites.

We provide custom paired sgRNA design for use in conjunction with Cas9 Nickase. Tell us your target gene by emailing [email protected].
Tools for gRNA Design

There are many tools available to help scientists in the designing process of gRNAs. Here we will highlight two of such tools: Chop Chop Harvard and CRISPR Design.

1. Chop Chop Harvard

Once a particular nucleotide sequence or accession number of a gene of interest is entered into Chop Chop Harvard (Figure 5), the software analyses the sequence and identifies all possible 20bp sequences which are immediately followed by the PAM sequence (5’-NGG). It then scores the gRNA according to a pre-set code, which looks at GC content and off target sites, and arranges them from best scoring to lowest scoring. For more information on a particular gRNA, simply click on it on the gRNA sequence. This will open a page that shows the potential off-target sites for that particular gRNA and even provides primer sequences that could be used to screen these off-target sites for potential mutations through the Surveyor assay. For more information on the Surveyor assay read our CRISPR Cas9 - Screening and Validation article (includes a detailed video on how to use Chop Chop Harvard see gRNA Design).

CRISPR Cas9 - Chop Chop Harvard

Figure 5 – The Chop Chop Harvard results page highlighting the ranking, exon position, and potential off target site counts.

2. CRISPR Design

The main advantage of using CRISPR Design is its ability to provide detailed information on Off-Target sights of all potential gRNA. It BLASTS every gRNA sequence and provides a detailed report about its off-target positions and the number of mismatches with the designed gRNA. This software is also superior when designing two gRNAs for paired nickase activity as it will automatically find two gRNAs that are within close proximity to one another.

However, the major drawback of this software is that it can only analyse 500 base pairs of sequence at a time. Therefore, we suggest the use of Chop Chop Harvard for picking potential gRNA sequences and then recommend further analysis with CRISPR Design tool.

Going through tons of designing work? Try our CRISPR sgRNA Library Service to knockout up to 100 target genes with a single custom targeted sgRNA pool against gene families and pathways.
CRISPR Webinar

CRISPR Experimental Design Tool
  • RNA-Guided Human Genome Engineering via Cas9. Mali, Prashant, et al. 6121, January 3, 2013, Science, Vol. 339, pp. 823-826.
  • CRISPR-Cas system for editing, regulating and targeting genomes. Sander, Jeffry D and Joung, J Keith. 4, April 2014, Nature Biotechnology, Vol. 32, pp. 347-355.
  • Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Fu, Yanfang, et al. Sep 1 2014, Nature Biotechnology, Vol. 32, pp. 279-294.
  • DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, Patrick D, et al. July 21, 2013, Nature Biotechnology, Vol. 31, pp. 827-832.
  • Genome engineering using the CRISPR-Cas9 system. Ran, F Ann, et al. 11, October 24, 2013, Nature Protocols, Vol. 8, pp. 2281-2308.