New England Biolabs, M0669M, EnGen® SpRY Cas9

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: Why is it called EnGen SpRY Cas9? A: The name SpRY is derived from S. pyogenes and the preference for 5´-NRN-3´ over 5´-NYN-3´ PAMs in vivo (R denotes purines (A or G) and Y denotes pyrimidines (C or T)). Nonetheless, SpRY Cas9 displays essentially no PAM preference in vitro. Q: What is the difference between EnGen SpRY Cas9, EnGen Spy Cas9 HF1 (NEB #M0667) and EnGen Spy Cas9 NLS (NEB #M0646)? A: EnGen Spy Cas9 NLS (NEB #M0646) encodes the wild-type Cas9 amino acid sequence whereas EnGen Spy Cas9 HF1 (NEB #M0667) encodes four point mutations in the Cas9 amino acid sequence (N497A, R661A, Q695A, Q926A) for reduction of off-target cleavage. EnGen SpRY Cas9 encodes 11 point mutations in the Cas9 amino acid sequence (A61R, L1111R, D1135L, S1136W, G1218K, E1219Q, N1317R, A1322R, R1333P, R1335Q, T1337R) for a relaxed PAM requirement. Q: How do I design a single guide RNA for use with EnGen SpRY Cas9? A: EnGen SpRY Cas9 utilizes the same single guide RNA scaffold as other S. pyogenes Cas9s: 5´-NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3´ N’s represent the 20 nucleotide sequence specific to the DNA target A synthetic single guide RNA may be purchased from an oligonucleotide synthesis company Alternatively, if higher yields are required, the NEB EnGen sgRNA Synthesis Kit, S. pyogenes (NEB #E3322) may be purchased The following figure illustrates how to design guide(s) for a specific DNA cut site: For every desired double stranded break on the DNA target, two single guide RNAs are available (targeting either strand of the DNA) when using EnGen SpRY Cas9. Q: How do I dilute the EnGen® SpRY Cas9 enzyme to 1 μM for in vitro reactions? A: If dilution is necessary, dilute the provided 20 μM enzyme solution 1:20 with 1X NEBuffer™ r3.1 before setting up in vitro reactions. Q: Why do I observe incomplete digestion/editing with EnGen® SpRY Cas9? A: Incomplete digestion may be due to the following factors: Suboptimal 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: DNA target. For plasmid linearization, higher ratios of nuclease/sgRNA may be required. 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™ r3.1 included with the enzyme. Q: Is it necessary to clean up my reaction before using the digestion products with NEBuilder? A: A clean up of the SpRY digest prior to cloning with NEBuilder is not required, but may improve results if performed. Q: Where is the nuclear localization signal on EnGen SpRY Cas9 located? A: EnGen SpRY Cas9 contains a nuclear localization signal located on the C-terminus of the protein. Q: Which nuclear localization signal is fused to EnGen SpRY Cas9? A: EnGen SpRY Cas9 contains Simian virus 40 (SV40) T antigen nuclear localization signal on the C- terminus of the protein. Q: Does NEB provide plasmids for guide RNA cloning? A: We do not distribute plasmids for sgRNA cloning, but we recommend using Addgene if you want to obtain sgRNA plasmids. DNA templates containing a T7 promoter and encoding sgRNA can be used with the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB #E2050). We recommend using the EnGen sgRNA Synthesis Kit, S. pyogenes (NEB #E3322S), which utilizes target-specific DNA oligos designed by the user and provides a quick method for transcribing high yields of sgRNA in a single 30-minute reaction. 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 we can offer this enhancement at the same price