New England Biolabs, M0654T, EnGen® Sau Cas9

The S size of this product was discontinued on July 31, 2023. M0654T will remain available for purchase. Related Categories CRISPR/Cas Nucleases Specification Reaction Conditions 1X NEBuffer™ r3.1 Incubate at 37°C NEBuffer™ r3.1 100 mM NaCl 50 mM Tris-HCl 10 mM MgCl2 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Storage Buffer 20 mM Tris-HCl 300 mM NaCl 0.1 mM TCEP 50% Glycerol pH 7.5 @ 25°C Heat Inactivation 65°C for 5 minutes FAQ Q: How can the genome editing efficiency be determined following EnGen® Sau Cas9 (NEB #M0654) transfections or microinjections? A: T7 Endonuclease I (NEB #M0302) or the EnGen Mutation Detection Kit (NEB #E3321S) can be used to determine editing events generated by Sau Cas9 RNP transfections or microinjections. Other methods, including amplicon sequencing, can also be used for analysis of editing. Q: Which nuclear localization signal is fused to EnGen® Sau Cas9? A: EnGen Sau Cas9 contains Simian virus 40 (SV40) T antigen nuclear localization signals (NLS) at both the N- and C-termini of the protein. Q: Can the EnGen® sgRNA Synthesis Kit (NEB #E3322S) be used to synthesize sgRNAs specific for Sau Cas9? A: No. The EnGen sgRNA Synthesis kit is specific for S. pyogenes (Spy). Q: What is the sequence for a typical guide RNA specific for EnGen® Sau Cas9? A: A typical Sau guide RNA is ~100 nucleotides long with a target specific sequence that is 20-22 nucleotides long (shown as N’s in the following example) and an Sau-specific sequence that will be the same for all guide RNAs: 5′NNNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUU3′ The 20-22 nucleotide region will vary based on the target DNA, but will never include the PAM (NNGRRT, N = any, R = A or G) sequence. The PAM sequence is located on the target DNA, just 3´ to the non-targeted DNA strand in the target region. An example can be found below. The bottom strand of the dsDNA in this example is the target strand. The PAM (NNGRRT, or in this example ACGGAT) is found on the non-targeted strand just 3´ to the target sequence. Q: How can I synthesize a sequence-specific sgRNA for experiments using EnGen® Sau Cas9? A: sgRNAs to be used with EnGen Sau Cas9 can be synthesized in vitro using the HiScribe® T7 High Yield RNA Synthesis Kit (NEB #E2040) or the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB #E2050). Double-stranded DNA templates can be either PCR products, synthetic double-strand DNA templates or linearized plasmids. More detailed design recommendations can be found in the protocols section. Alternatively, a synthetic sgRNA could be ordered from an RNA synthesis company. Q: Why do I observe incomplete digestion with EnGen® Sau Cas9 and my target-specific Sau sgRNA in vitro? A: The efficiencies of sgRNAs vary depending on the specific target sequences. We recommend using an on-line tool (which use certain factors such as GC content) to select the best target site within your DNA of interest. We also recommend purification of the dsDNA target and verifying the integrity of the sgRNA on a denaturing gel. If sgRNA efficiency is low, increasing the molar ratio of sgRNA:Sau Cas9:target DNA to 10:10:1 or higher may improve DNA cleavage. We also recommend using the supplied NEBuffer™ 3.1 for best results. Q: How do I dilute the enzyme to 1 μM for in vitro reactions? A: If planning to dilute the EnGen Sau Cas9 (NEB #M0654T) for in vitro digestion of DNA, the enzyme can be diluted to 1 μM in 1X rNEBuffer™ r3.1 prior to the reaction assembly and used immediately. If the 1 μM dilution will be stored at -20°C, it should be diluted in the enzyme storage buffer: 20 μM Tris-HCl, 300 μM NaCl, 0.1 μM TCEP and 50% glycerol (pH 7.5 @ 25°C). Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price.