Single-guide RNA, or sgRNA, is an engineered molecule that directs the CRISPR-Cas9 gene-editing system to a specific DNA sequence. It functions as a programmable guide, telling Cas9 precisely where to cut. This technology was adapted from a natural defense mechanism in bacteria. The system’s two main components are the sgRNA for specificity and the Cas9 enzyme for DNA cleavage.
The Engineered Components of sgRNA
The sgRNA is a synthetic fusion of two natural bacterial RNA molecules: the CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA). Scientists combined these into a single molecule to simplify its use in the lab. This single-guide format is now the most common approach for researchers using CRISPR technology.
The sgRNA’s structure has two functional parts. The first is a guide sequence of approximately 20 nucleotides derived from the crRNA. This portion is custom-designed to be complementary to the target DNA, giving the system its precision.
The second part is the tracrRNA-derived scaffold, which forms a structure that acts as a handle for the Cas9 protein. By binding to this scaffold, the Cas9 protein is positioned to interact with the target DNA. Fusing the guide sequence and scaffold into one molecule was an important step in making CRISPR a widely accessible tool.
Guiding the Molecular Scissors
The primary function of sgRNA is to direct the Cas9 protein, or “molecular scissors,” to a precise location within a cell’s genome. Once inside a cell, the sgRNA and Cas9 protein assemble into a ribonucleoprotein complex. This complex then scans the DNA for a sequence matching the sgRNA’s guide sequence.
For Cas9 to bind and cut DNA, it must first recognize a short sequence known as a Protospacer Adjacent Motif (PAM). The PAM sequence is not part of the target sequence but is located on the DNA strand just downstream from it. The most commonly used Cas9 from Streptococcus pyogenes recognizes the sequence NGG, where N can be any nucleotide.
After the complex binds to a PAM sequence, the sgRNA’s guide sequence unwinds the adjacent DNA double helix and pairs with its complementary strand. This pairing confirms the correct location and induces a change in the Cas9 protein, activating it to cut both DNA strands. The cut is typically made three nucleotides upstream of the PAM sequence.
Designing a Custom Guide
Creating a custom sgRNA begins with identifying a target gene. Researchers then use computational software to design the 20-nucleotide guide sequence. These tools require inputs like a DNA sequence, gene name, or genomic location, along with the species being studied.
A key part of the design process is ensuring the guide sequence is unique to the target site to avoid off-target effects. Bioinformatics algorithms analyze the organism’s genome to find other DNA locations similar to the target sequence. The software then scores potential guide sequences on their predicted on-target effectiveness and off-target binding likelihood.
Tools like CHOPCHOP and E-CRISP rank potential guide sequences using different methods, such as empirical data or user-defined penalties for mismatches. This careful planning aims to maximize editing activity at the desired location while minimizing unintended cuts elsewhere in the genome.
Applications in Science and Medicine
The ability to design custom sgRNAs has applications in both scientific research and medicine. In basic research, scientists use sgRNAs to “knock out” genes in cell cultures or model organisms like mice and fruit flies. By observing the results, they can deduce the function of the inactivated gene.
In medicine, sgRNA-based therapies are being developed to correct mutations that cause inherited diseases like sickle cell anemia. For this condition, researchers designed sgRNAs to target the gene for fetal hemoglobin, which is normally inactive after birth. Reactivating this gene can compensate for the defective adult hemoglobin.
This technology is also being investigated for treating genetic forms of blindness by correcting mutations in retinal cells. Beyond therapeutics, sgRNAs are used in diagnostic tools. These CRISPR-based diagnostics can be programmed to detect specific DNA or RNA sequences from viruses or cancer, offering a new platform for disease detection.
Delivering the Guide and Avoiding Errors
A challenge in using CRISPR-Cas9 is delivering the sgRNA and Cas9 components into target cells. Therapeutic applications often use a vector, such as adeno-associated viruses (AAVs), to carry the genetic instructions. AAVs are used because they do not cause disease and efficiently enter many cell types.
Non-viral methods are also being developed, with lipid nanoparticles (LNPs) as a leading alternative. The sgRNA and Cas9 are encapsulated in a small fat bubble that fuses with the cell membrane to release its contents. This is the same delivery technology used in some mRNA vaccines.
Even with a well-designed sgRNA, there is a risk of “off-target effects,” where Cas9 cuts DNA at unintended locations with a similar sequence. While bioinformatics tools minimize this risk during design, researchers are developing other strategies to improve specificity. These include using high-fidelity Cas9 variants engineered for precision and modifying the sgRNA to enhance its stability and reduce immune responses.