New England Biolabs, M0650T, EnGen® Spy Cas9 Nickase

Related Categories CRISPR/Cas Nucleases Specification Reaction Conditions 1X NEBuffer™ r3.1 Incubate at 37°C 1X 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 300 mM NaCl 10 mM Tris-HCl 0.1 mM EDTA 1 mM DTT 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 5 minutes FAQ Q: What is the difference between EnGen® Spy Cas9 Nickase (NEB #M0650) and EnGen Spy Cas9 NLS (NEB #M0646)? A: EnGen Spy Cas9 Nickase contains a single point mutation (D10A) that inactivates the RuvC nuclease domain, but leaves the HNH nuclease domain intact, rendering the enzyme a nickase. EnGen Spy Cas9 NLS has both nuclease domains active and is able to cleave both strands of dsDNA. Q: Which nuclear localization signal is fused to EnGen® Spy Cas9 Nickase? A: EnGen® Spy Cas9 Nickase contains Simian virus 40 (SV40) T antigen nuclear localization signal (NLS) on the N- and C- termini of the protein. Q: Why does binding efficiency differ between two different guide RNAs? A: Binding efficiency may be influenced by guide RNA design. Verify the sequence and design of the guide RNA transcription template. It may also be influenced by guide RNA quality. Verify the integrity of the guide RNA by gel electrophoresis. Q: Does NEB offer synthetic sgRNAs or plasmids for cloning? A: NEB does not offer sgRNA synthesis services or plasmid for sgRNA cloning. For cloning we recommend visiting Addgene to obtain sgRNA plasmids. For quickly transcribing sgRNAs without the need for cloning or PCR, we recommend the EnGen sgRNA Synthesis Kit, S. pyogenes (E3322S). Alternatively, oligonucleotide templates encoding an sgRNA under a T7 promoter (PCR products or plasmids) can be used to transcribe sgRNA using an in vitro transcription kit such as NEB's HiScribe T7 Quick High Yield RNA Synthesis Kit (E2050). Q: How should offset sgRNAs be oriented for efficient modification using EnGen® Spy Cas9 Nickase? A: Reports on genome modification using Cas9 Nickase (D10A or H840A) suggest efficient modification occurs when sgRNAs are oriented in a tail-to-tail (i.e., 5´ – 5´) orientation and when separated by 10 and 30 bp. Cleavage in this orientation results in 5´ overhanging ends. Editing was detectable with overlapping sgRNA pairs and with pairs at greater distances apart (≥40 bp), albeit with reduced efficiency (Ran et al. (2013) Cell, 155, 479-480). We recommend trying an assortment of sgRNA pairs to identify a set that provides the desired editing efficiency. Q: Why do I observe incomplete digestion? A: Incomplete digestion may be due to the following factors: Incorrect ratio of Cas9 Nuclease to guide RNA, and target site- For complete digestion we recommend a 10:10:1 or higher molar ratio of Cas9 Nuclease: guide RNA : target site. Suboptimal sequence of the guide RNA- Verify the sequence and design of the guide RNA. Poor quality guide RNA- Verify the integrity of the guide RNA by gel electrophoresis. Suboptimal buffer- Please use the NEBuffer 3.1 included with the enzyme. Q: How do I dilute the enzyme to 1 μM for in vitro reactions? A: If planning to use the higher concentration enzyme for in vitro digestion of DNA, the enzyme can be diluted to 1 μM in 1X NEBuffer™ r3.1 and used immediately. The 1 μM dilution in 1X NEBuffer r3.1 should not be frozen. If the 1 μM dilution will be stored at -20°C, it should be diluted using Diluent B (with rAlbumin) (NEB #B8533S): 300 μM NaCl, 10 μM Tris-HCl, 0.1 μM EDTA, 1 μM DTT, 500 μg/ml Recombinant Albumin and 50% glycerol (pH 7.4 @ 25°C) prior to the reaction assembly. 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.