What Is Guide RNA (gRNA)? Definitions & Applications

What Is Guide RNA (gRNA)? Definitions & Applications

What Is Guide RNA (gRNA)?

Gene editing, also known as genome engineering, is a revolutionary technique that modifies DNA to treat disease and improve human health.

Among such techniques is the CRISPR-Cas system, standing for clustered regularly interspaced short palindromic repeats. It is composed of two components:

  • Guide RNA (gRNA): A specific RNA sequence that recognizes the region of interest in the target DNA. It binds with the Cas9 protein and directs it to the target site to perform the modification process.

The CRISPR gRNA is itself composed of two units:

  • CRISPR RNA (crRNA): Composed of 17-20 nucleotides sequences that are complementary to the target DNA sequence, and also possesses a spacer flanked region due to repeat sequences.
  • tracrRNA: Acts as a binding scaffold for the Cas nuclease.
  • CRISPR-associated (Cas) nuclease: A non-specific endonuclease enzyme, which is directed at the target sequence by CRISPR guide RNA to make the double-strand break.  

Even though Cas nuclease has been isolated from different bacteria, the most commonly used one, SpCas9, is isolated from Streptococcus pyogenes.

There’s also another variant of guide RNA, which consists of a single RNA molecule and is called single-guide RNA (sgRNA). It has a custom-designed short crRNA sequence conjoined with the scaffold tracrRNA sequence to facilitate the process of cleavage.

Figure: An illustration of the formation of sgRNA and Cas9 complex.

In this article, we will review the working mechanism of the gRNA in the CRISPR-Cas9 system, gRNA design in labs for lab assays, and altering the genome sequence of organisms using gRNA for research studies.

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How Does gRNA Work?

gRNA works in combination with the CRISPR-CAS9 system. The two parts of the system, crRNA and tracrRNA work in harmony to carry out the genome editing process. The gRNA’s tracrRNA identifies cas9 proteins and binds with them. Then, the cas9 proteins obtain the sequence information from crRNA and walk toward the target site.

After identifying the target site, crRNA binds to the sequence and stabilizes the DNA-RNA complex. The cas9 protein identifies the PAM (protospacer adjacent motif, usually 2-6 base pairs in length) sequence, 5’-NGG-3’ region, and starts the cleavage process upstream to the binding site of crRNA.

Figure: An illustration of the role of gRNA in the CRISPR-Cas9 system.

gRNA in Protists

In the mitochondria of protists, Leishmania tarentolae, a post-transcriptional RNA editing procedure occurs.

The protists have minicircle and maxicircle DNA. When the majority of maxicircle (having 16-17 kilobase pairs of the coding region, encoding some gRNA as well) transcripts or mRNA can’t proceed to protein synthesis due to frameshift mutation, the guide RNA corrects the mutation post-transcriptionally by the deletion and insertion of uridine residues at specific locations.  

Designing gRNA

Scientists modify the wild-type gRNA sequence in labs to test its efficacy in editing nucleic acids and developing treatments for many life-threatening diseases.

Currently, CRISPR library design tools are available to help researchers easily design and optimize multiple gRNA sequences with increased editing efficiency, validation, and reduced off-target effects.

The gRNA can be designed in vitro or in vivo using template DNA and custom-designed crRNA fused with the scaffold tracrRNA sequence.

The targeting specificity of the CRISPR-Cas9 system mainly depends on the 20 nucleotide sequence present at the 5’-end of the gRNA. The target sequence usually precedes the protospacer adjacent motif (PAM), which follows the targeted cleavage region.

When the gRNA is formed (by joining the crRNA and tracrRNA), it binds with the target site and assembles a ribonucleoprotein (RNP) complex with the Cas9 protein to initiate a double-strand break at a locus about 3-nucleotide upstream of PAM.

Before designing a gRNA, ensure that the GC content of the gRNA is between 40-80% and the length of the gRNA sequence should be around 17-24 base pairs to minimize the off-target effects.

Predicting gRNA Efficacy

There’s no universal technique available to select the right gRNA for your experiments. However, the factors that impact its efficacy and accuracy include:

  • The method used to produce a transfection-ready guide RNA, such as in vitro transcription, synthetic method, or lentiviral delivery technique.
  • The dynamic aspect of target nucleic acid, such as accessibility to the target site due to chromatin arrangement or status.

While working with the transcriptional modulation and knockout approaches, it’s recommended to examine a spectrum of gRNA designs across a gene and not only conclude based on one test.

What Is gRNA Used For?

Recombinant gRNA is used in labs along with other reagents to perform a range of laboratory assays including classic PCR, multiplex PCR, whole-genome sequencing, cloning, development of cell lines, primers, and recombinant plasmid DNA, and mismatch assay for DNA modification, mutagenesis, and gene expression studies.

Below are some of the applications involving gRNA uses.

Genome Editing

CRISPR-Cas system is an efficient, simple, and versatile tool used in labs for genetic modification.

After the Cas9 protein binds to the target sequence, it makes a double-strand break upstream to the PAM sequence. The break can be repaired in two ways:

  • Non-homologous end joining (NHEJ): An error-prone method which is utilized when no donor DNA is present. It helps to produce an indel resulting in an effective knockout of protein functions.
  • Homology-directed repair (HDR): A method put into function when donor DNA sequences are available. It has a role in creating an accurate knock-in of the target gene.

Genome editing using CRISPR-Cas is effective and simple, because of which it’s more approachable by labs—even by those not having expertise in molecular biology—for genome engineering, molecular, and functional analysis.

Gene Activation and Silencing

CRISPR-Cas9 system is one of the most predominant techniques used by researchers for gene activation, gene expression, and gene inhibition studies. By modifying the Cas9 protein, it can be used to mechanically regulate the targeted gene activity.

Researchers have created a modified dCas9 protein, which lacks cleavage activity but possesses DNA binding affinity. It can easily be fused with the transcriptional activator (CRISPRa) and inhibitor (CRISPRi), leading to control of the expression of a gene.

Moreover, it can also be fused with fluorescent markers, such as Green Fluorescent Proteins (GFP), to detect the location of a specific gene of interest.

Figure: An schematic diagram of the mechanism of knock-out and knock-in using CRISPR-Cas.

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gRNA is an integral part of the CRISPR-Cas system, where it forms a complex with the Cas9 protein to create breaks in nucleic acids.

It’s composed of two components, crRNA and tracrRNA, that both help in fulfilling the gRNA functions. Scientists in labs modify the sequences of wild-type crRNA and use it as a tool for their research studies including gene modification, gene activation and silencing, and genome engineering.

Apart from being a simple and versatile technique, CRISPR-Cas is an expensive technique as well. Thus, if you’re working on high-throughput experiments, we recommend proceeding with high-tech lab equipment. Getting proper equipment helps you to save time by eliminating the requirement of repeating experiments and potentially wasting reagents.

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