CRISPR/Cas9 is a technique in which RNA-directed Cas nuclease to make specific DNA modifications to targeted genes, which is an acquired immune defense mechanism evolved by bacteria and archaebacteria in response to attacks by phages and foreign plasmids. In this system, the Cas9 nuclease is guided with artificially optimized single guide RNA (sgRNA) to cut double-stranded DNA (dsDNA) at the target site causing DNA breaks. DNA is repaired with the non-homologous end joining or Homology directed repair mechanism in vivo, resulting in frameshift mutation, replacement or deletion of genes. Hifair™ Precision sgRNA Synthesis Kit uses a T7 RNA polymerase mixture to efficiently transcribe spCas9 sgRNA. The kit contains a specific sequence that is recognized by the Cas9 protein and acts as a scaffold. A portion of the sequence can be partially overlapped with the target specific sequence designed by the user. The complementary strands are filled by DNA polymerase to generate dsDNA templates for transcription. Using its supplied reagent and user-designed specific DNA sequences, the kit can obtain 20-100 μg of functional sgRNA within 4 hours in a single tube reaction. Features High Synthesis Yield – Achieve 20–100 µg sgRNA in a single-tube reaction within just 4 hours. Broad Applicability – Consistently high yields across various sgRNA sequences and target types. Exceptional Purity – Produces sgRNA with a distinct single band, ensuring minimal by-products. Proven Functional Activity – Synthesized sgRNA efficiently guides Cas9-mediated DNA cleavage. Applications CRISPR/Cas9 Genome Editing; In Vitro Cleavage Assays; Functional Genomics Studies; Gene Therapy Research; Components Components No. Name 11355ES05 11355ES25 11355ES50 11355ES60 11355-A 10×sgRNA Enzyme Mix 10 μL 50 μL 100 μL 200 μL 11355-B 10×sgRNA Reaction Buffer 10 μL 50 μL 100 μL 200 μL 11355-C 2×Canace Enzyme Mix 62.5 μL 312.5 μL 625 μL 1.25 mL 11355-D Scaffold Template 6.25 μL 31.25 μL 62.5 μL 125 μL 11355-E NTPs (25 mM each) 40 μL 200 μL 400 μL 800 μL 11355-F Control sgRNA Oligo (10 μM) 2 μL 10 μL 20 μL 40 μL Shipping and Storage This product should be stored at -25 ~ -15℃ for 2 years. Figures 1. High Synthesis Yield Figure 1. sgRNA yield at different reaction times. Using the HifairTM Precision sgRNA Synthesis Kit, a single-tube reaction can produce 20–100 µg sgRNA within 4 hours, fully meeting the requirements of genome editing applications. 2. Broad Compatibility – Achieves high yields across various sgRNA types. Figure 2. Synthesis yield of different sgRNA types. Ten different sgRNAs were synthesized in parallel using the HifairTM Precision sgRNA Synthesis Kit. Results show that the kit consistently delivers high yields across multiple sgRNA types, meeting the needs of diverse gene-editing applications. 3. High Purity – Synthesized sgRNA exhibits a single, distinct band with minimal impurities. Figure 3. Quality assessment of synthesized sgRNA (Agilent 2100). sgRNA synthesized using the HifairTM Precision sgRNA Synthesis Kit was analyzed on an Agilent 2100 system. The results show a distinct single peak with no secondary peaks, indicating high purity. 4. High Activity – Effectively guides Cas9 nuclease to cleave target DNA with high efficiency. Figure 4. Cleavage efficiency of synthesized sgRNA. sgRNA synthesized using the HifairTM Precision sgRNA Synthesis Kit was combined with Cas9 nuclease for in vitro cleavage of target DNA. Agarose gel electrophoresis revealed that the synthesized sgRNA effectively guided Cas9 to cleave at the specific target site, producing DNA fragments of the expected size. Documents: Safety Data Sheet 11355_MSDS_HB250625 Manuals 11355_Manual_Ver.EN20240710
