What is CRISPR Technology?
CRISPR/CasX Technology (Clustered Regularly Interspaced Short Palindromic Repeats), derived from bacteria, has been a cornerstone of genetic engineering in the therapeutic industry for decades. The technology is versatile with its application, spanning across various fields, from agriculture to medicine, and enabling scientists to modify DNA sequences with remarkable accuracy. The most well-known system is CRISPR/Cas9 with its approach in utilizing a guide RNA (gRNA) to target specific genomic locations, directing the Cas9 enzyme to make precise cuts in the DNA. Despite its immense potential, CRISPR technology still faces limitations, such as immune responses triggered by the transport system to the target site, off-target effects, and incomplete edits. Further research is needed to address these challenges for therapeutic applications.
What is the role of gRNA/sgRNA in the CRISPR system?
gRNA and sgRNA are being called synonymously in the field since they are both referred to the same mechanism of action but in different formats. Let’s us explain the difference:
In traditional CRISPR systems, guide RNA (gRNA) is composed of two RNA molecules: CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). These two RNA molecules must bind together to form the functional guide RNA. The crRNA contains a specific sequence complementary to the target DNA region to help guide the Cas protein to the correct location for editing. TracrRNA acts as a scaffold to stabilize the complex formed between the crRNA and the Cas protein, aiding in the recognition and cleavage of the target DNA.
Single-guide RNA (sgRNA) is a chimeric RNA molecule that combines the functions of crRNA and tracrRNA, streamlining the process by eliminating the need for separate components. sgRNA is more popular due to its simplified format, making it easier to design and implement in experiments.
There is a rising interest in utilizing long chain sgRNA due to its enhanced specificity and reduced off-target effects [1,2]. Synoligo is ready to meet the increasing demand for long chain sgRNAs, catering to various lengths, scales, and analytical requirements.
What is Synoligo’s capability in synthesizing ultra-long oligos?
Yield, purity, and accuracy become more challenging as the length of the oligo increases. However, our team of experts routinely synthesize ultra-long oligos at lengths of ~150 and ~180 nucleotides using our optimized long oligo synthesis platform. We are among the few companies capable of offering long-mers with high purity through chemical synthesis rather than enzymatic methods.
With our state-of-the-art synthesis capabilities, we can easily produce long oligos up to 200 nucleotides with modifications, tailored to our customers’ specific needs. Additionally, we can guarantee low endotoxin level less than 0.01EU/mg, even for the most challenging lipid-modified oligos. Whether you’re exploring gene therapy, studying disease mechanisms, or engineering sustainable crops, we’ve got you covered. Contact us for more information!
References:
- Matson AW, Hosny N, Swanson ZA, Hering BJ, Burlak C. Optimizing sgRNA length to improve target specificity and efficiency for the GGTA1 gene using the CRISPR/Cas9 gene editing system. PLoS One. 2019 Dec 10;14(12):e0226107. doi: 10.1371/journal.pone.0226107. PMID: 31821359; PMCID: PMC6903732.
- Guo C, Ma X, Gao F, Guo Y. Off-target effects in CRISPR/Cas9 gene editing. Front Bioeng Biotechnol. 2023 Mar 9;11:1143157. doi: 10.3389/fbioe.2023.1143157. PMID: 36970624; PMCID: PMC10034092.