The often overlooked sgRNA is the key to the success of CRISPR gene editing experiments!
On October 7, local time in Sweden, the 2020 Nobel Prize in Chemistry was awarded to French scientist Emmanuelle Charpentier and American scientist Jennifer A. Doudna in recognition of their "development of a method for genome editing" and their elaboration and development of CRISPR gene editing technology.
CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats Cas9) is a system composed of clustered regularly interspaced short palindromic repeats and Cas9 protein, which is the immune system of bacteria to resist foreign viral infection. Modern biology has widely used the CRISPR/Cas9 system in the field of gene editing . It is the most important gene modification technology after zinc-finger nuclease (ZFN) and transcription activator- like effector nuclease ( TALENs). It can be widely used in gene knock-in, gene knock-out, gene activation, gene silencing, epigenetic modification and 3D gene structure change [1].
.jpg@webp)
Figure 1. The three stages of the CRISPR/Cas bacterial adaptive immune system: acquisition, crRNA biogenesis and interference of viral DNA [2]
Applications of CRISPR/Cas9
1. Determination and verification of drug targets2. Analysis of upstream and downstream regulatory mechanisms of gene circuits3. Analysis of metabolic pathway regulation mechanisms4. Analysis of the mechanism of action of LncRNA
The CRISPR/Cas9 gene editing system is a sgRNA containing single-stranded RNA that uses a sequence complementary to the 5' end of the sgRNA to guide the Cas9 protein to the target DNA site. Cas9 Nuclease recognizes the target DNA sequence in a PAM-dependent manner and initiates DNA cutting at a specific site 3bp upstream of the PAM region . The double-strand breaks generated by Cas9 Nuclease can be repaired by NHEJ (Non-homologous end joining) or HDR (Homology directed repair). NHEJ repair usually leads to indel mutations and gene inactivation, while HDR can achieve high-fidelity and precise genome editing when a donor template is provided . Since organisms have self-repair mechanisms, as cells replicate, divide and repair the incision, mismatches, frameshift mutations, and deletion mutations may occur during the repair process, and finally mismatched homozygotes are screened.
The CRISPR/Cas9 gene editing system is a sgRNA containing single-stranded RNA that uses a sequence complementary to the 5' end of the sgRNA to guide the Cas9 protein to the target DNA site. Cas9 Nuclease recognizes the target DNA sequence in a PAM-dependent manner and initiates DNA cutting at a specific site 3bp upstream of the PAM region . The double-strand breaks generated by Cas9 Nuclease can be repaired by NHEJ (Non-homologous end joining) or HDR (Homology directed repair). NHEJ repair usually leads to indel mutations and gene inactivation, while HDR can achieve high-fidelity and precise genome editing when a donor template is provided . Since organisms have self-repair mechanisms, as cells replicate, divide and repair the incision, mismatches, frameshift mutations, and deletion mutations may occur during the repair process, and finally mismatched homozygotes are screened.
.jpg@webp)
Figure 2. An overview of CRISPR/Cas9 mediated genome editing [3]
The keys to the success of the CRISPR/Cas9 system include:
1) How to reduce and avoid off-target effects: using nick-form Cas9 plus double-stranded sgRNA2) Selection of sgRNA target: The Cas9 Nuclease cutting position is within 30 bp of the site to be modified, and the Z difference is kept within 50 bp. 3) sgRNA quality: sgRNA activity is one of the key factors affecting KI, so its activity needs to be verified in advance.4) Selection of Cas9 Nuclease form: Shortening the duration of Cas9 protein in cells can avoid continuous cutting and off-target. It is recommended to use Cas9 in mRNA or protein form. If plasmid is used, its purity and concentration must be ensured (recommended to be greater than 1μg/μl).
After meticulous research and development, Yeasen Biotechnology has developed an sgRNA in vitro synthesis kit that uses the reagents it provides and the specific DNA sequence designed by the user to obtain 20-100 μg of functional, high-quality sgRNA within 4 hours of a single-tube reaction . It has the characteristics of high synthesis efficiency, good quality, and high purity.
Hifair ® Precision sgRNA Synthesis Kit Test Data
A higher concentration of sgRNA is related to the success or failure of the editing effect of the CRISPR/Cas9 system, so only by adding a high concentration of sgRNA can the editing of genes be better. The sgRNA in vitro synthesis kit developed by Yisheng uses the reagents it provides and the specific DNA sequence designed by the user to obtain 20-100μg in a single tube reaction within 4 hours, which fully meets the needs of gene editing.
.jpg@webp)
Figure 3. The amount of sgRNA synthesized at different times
The sgRNA Synthesis Kit must not only meet the requirements of yield, but also achieve a fairly high yield for the synthesis of different types of sgRNAs, so as to meet the editing needs of different types of genes and expand the application scope of the kit. After synthesizing multiple types of sgRNAs, the yield of Yisheng sgRNA Synthesis Kit is also very high, which can meet the needs of experiments, and therefore can adapt to the editing needs of multiple genes.
.jpg@webp)
Figure 4. Synthesis amount of different types of sgRNA
Among the key factors that affect the success or failure of the editing effect of the CRISPR/Cas9 system, the quality of the synthesized sgRNA is also a key link. Because the purity (i.e. quality) of the synthesized sgRNA is not good, it will have a great impact on the efficiency of editing. The sgRNA synthesized by Yisheng sgRNA Synthesis Kit was measured by Agilent 2100, and its bands were obvious, single, and high in purity , indicating that it is of high quality and will greatly improve the subsequent editing efficiency.
.jpg@webp)
Figure 5. Quality of synthetic sgRNA (measured by Agilent 2100)
Finally, whether the synthesized high-quality sgRNA is active requires field testing to find out whether it is active and whether it can be combined with Cas9 Nuclease to achieve the purpose of editing genes. The sgRNA synthesized by Yisheng sgRNA Synthesis Kit is combined with Cas9 Nuclease to cut the target DNA in vitro. Agarose gel electrophoresis detection found that the synthesized sgRNA can effectively guide Cas9 Nuclease to cut at a specific site and produce a DNA fragment of a specific size, so it is active.
.jpg@webp)
Figure 6. sgRNA shearing efficiency effect diagram
Yeasen Biotechnology provides you with high-quality, cost-effective sgRNA synthesis kits, which will give your CRISPR/Cas9 scientific research and related gene editing applications a boost and contribute to the future health of mankind.