CRISPR/Cas Nucleases

Profiled for ribonucleoprotein (RNP) workflows, in vitro assays & cloning

NEB’s toolbox of Cas enzymes supports RNA-guided programmable cutting, binding and nicking of DNA. Distinct properties of engineered and wild-type Cas nucleases can be leveraged for many CRISPR applications. These applications include highly efficient genome editing in cells via ribonucleoprotein (RNP) direct delivery using Cas9 or Cas12a nucleases complexed with guide RNA, CRISPR-based screening assays, indel detection assays,and targeted dsDNA (i.e., plasmid) digests for cloning protocols. Cas9 Nuclease, Streptococcus pyogenes (Spy) (NEB #M0386) is the most commonly used, conventional RNA programmable endonuclease. The EnGen^® Cas nucleases are characterized for PAM sequence specificities, cleavage activities, temperature tolerances and the presence of nuclear localization signals that confer utilities to expand genome targeting or increase targeting specificity in CRISPR workflows.

Reduce CRISPR off-targeting

• EnGen Spy Cas9 NLS (NEB #M0646) contains dual nuclear localization signals for improved transport to the nucleus, which increases editing efficiency, enhancing the probability of on-target editing.
• EnGen Spy Cas9 HF1 (NEB #M0667) is a high-fidelity, quadruple substitution (N497A/R661A/Q695A/Q926A) variant of EnGen® Spy Cas9 NLS, with reduced off-targeting and dual nuclear localization signals, that is highly recommended for RNP delivery into cells and various in vitro assays.
• EnGen Spy Cas9 Nickase (NEB #M0650) can be used with different sgRNAs to target nicking of two sites in close proximity to generate dsDNA breaks with reduced off-targeting for in vivo or in vitro applications.
• EnGen Sau Cas9 (NEB #M0654) derived from Staphylococcus aureus, cleaves dsDNA, ~3 bases upstream of the longer PAM sequence 5′NNGRRT, enabling higher specificity and blunt ends.

Expand editing to new genome regions or leverage in vitro cleavage

• EnGen Lba Cas12a (Cpf1) (NEB #M0653), derived from Lachnospiraceae, has a 5´ TTTN PAM, with dsDNA cleavage activity in a range of 16 to 48°C, and additional non-specific, trans ssDNA cleavage that can be leveraged for in vitro assays.
• EnGen Seq1 Cas9 (NEB #M0668) is a dsDNA endonuclease, derived from Streptococcus equinus, that expands targeting regions with a  5´- NAGA -3´ PAM sequence.
• EnGen SpRY Cas9 (NEB #M0669) has a non-specific PAM (5´-NNN-3´ PAM) that broadens genome editing and nearly eliminates sequence constraints in vitro.  Precise cleavage in vitro streamlines large construct cloning workflows.

Enrich or visualize Cas RNP complexes

• EnGen Spy dCas9 (SNAP-tag^®) (NEB #M0652) is an inactive mutant that allows attachment of fluorophores, biotin, and several other conjugates, offering molecular tag flexibility for target enrichment or visualization.

Synthesize sgRNA for use with our Cas enzymes

Use S. pyogenes-derived Cas nucleases in conjunction with the EnGen sgRNA Synthesis Kit (NEB #E3322), or use our portfolio of RNA Synthesis and Modification solutions to generate sgRNAs compatible with alternative Cas nucleases.

A Toolbox of Cas Nucleases for CRISPR Workflows