Custom gRNA Synthesis for CRISPR/Cas9

What is gRNA?

gRNA (Guide RNA) is part of the CRISPR-Cas9 system. gRNA molecules have two components, a target-specific CRISPR RNA (crRNA) and a helper trans-activating crRNA (tracrRNA). gRNA imports Cas9 proteins into a specific gene locus by base-pairing between a 20-mer crRNA sequence and the target genomic sequence. Cas9 proteins into specific gene loci through base pairing between 20-mer crRNA sequences and target genomic sequences. The design, generation, and delivery of high-quality gRNAs is critical to the success of using the CRISPR-Cas9 system to knock down gene expression or to knock in specific mutations. BOC Sciences is committed to utilizing its expertise and experience to produce high-quality gRNAs tailored to meet the individual needs of our customers.

Fig 1. Synthetic gRNAs can be applied to a variety of CRISPR-Cas9 experimental approaches. (Kelley et al., 2016)

What is the Function of gRNA in CRISPR/Cas Technologies?

• Target Recognition

The gRNA sequence is designed to be complementary to the target site in the genome. When gRNA is expressed or introduced into a cell with Cas enzyme, it directs Cas enzyme to the specific DNA/RNA sequence of interest.

• Cas Enzyme Binding

gRNA forms a complex with Cas9 through base pairing interactions. Cas9 enzyme recognizes the target site based on the complementarity between the gRNA sequence and the target DNA/RNA sequence.

• Site-specific Cleavage or Modification

Once the gRNA-Cas complex binds to the target site, the Cas enzyme induces a double-strand break (DSB) in the DNA, depending on the specific CRISPR-Cas system used.

• DNA Repair Mechanism

DSBs generated by Cas enzyme trigger cellular DNA repair mechanisms that can be used to introduce specific genetic changes, such as gene knockouts, gene insertions, or gene replacements.

BOC Sciences' Custom gRNA Synthesis Services for CRISPR/Cas9

• gRNA Design

gRNA design is a critical step in CRISPR/Cas9 experiments as it determines the specificity and efficiency of genome editing. Consideration needs to be given to the selection of the target, which needs to fulfill the corresponding conditions.

(1) It should target the desired gene or genomic region to avoid off-target effects.

(2) Target sites with repetitive sequences or regions of low complexity are avoided as they may lead to off-target effects or inefficient editing.

(3) Preferably, select target sites within exons or regions known to be functionally significant.

In addition, parameters such as gRNA length, prototypical spacer adjacent motifs, need to be examined. BOC Sciences also offers a selection of predicted off-target sites, which can minimize off-target effects by selecting target sites with the fewest predicted off-target sites or no predicted off-target sites. Potential off-target effects can be assessed using tools such as CRISPR design software. Ensure that the gRNA does not form a stable secondary structure, especially at the 3' end, as it may interfere with the formation of the gRNA-Cas9 complex.

• Custom gRNA Synthesis for CRISPR/Cas9

After testing the design of the gRNA, the gRNA can be prepared using an in vitro transcription method. Of course, you can also request a custom product from us and we will customize your gRNA according to your requirements.

• Purification of gRNA

After gRNA is synthesized, it needs to be purified to remove any impurities or contaminants. After purification, the gRNA should be quantified to determine its concentration.

• Quality Control and Validation

It is important to validate the synthesized gRNA to ensure its quality and functionality. This can be accomplished through gel electrophoresis to confirm the correct size of the gRNA and through sequencing to verify sequence accuracy.

BOC Sciences is dedicated to oligo synthesis services based on our technology and Lab equipment, and we are constantly updating the types of services we offer, so stay tuned and we will provide you with the services you need.

Reference

1. Kelley M L, et al. Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing[J]. Journal of biotechnology, 2016, 233: 74-83.