Nuclease variants

Available nuclease variants

Note: These plasmids have been developed by groups outside Erasmus MC and before use a Material Transfer Agreement (MTA) has to be signed with the original depositor of the plasmid.


Cas9

Referred to as WT from S. Pyogenes, but has human codon optimization and added NLS sequences. Guide RNA can be cloned into the backbone in a single- or multiplex version where several guides targeting different genes can be combined. Also versions with a GFP fluorescent reporter or puromycin selection are available.

Cas9N

Nickase mutant of Cas9 (D10A mutant).

Cas9D

Kinase dead form of Cas9 and can only recruit CRISPR/Cas9 complex to the DNA without cleaving it. Used for enCHIP analysis to purify genomic regions of interest.

Cas9-HF1

High fidelity form of Cas9 with 4 mutations to prevent off target effects (except single bulges in the seed sequence), but retains on target nuclease activity.


Cas9-HF4

High fidelity form of Cas9 with 4 additional mutations to prevent off target effects with single bulges in the seed sequence recognition site, but with slightly reduced on target nuclease activity.

Cas9-VRQR-HF1

High fidelity form of Cas9 with additional mutations to alter the PAM sequence expanding the guide recognitions sites in the genome(recognizes NGAN).

eSpCas9

Enhanced form of Cas9 with similar function as the high fidelity form of Cas9, but from another source.

Cpf1

Alternative type II nuclease, recognizes a different PAM sequence (NTTT) and makes a staggered cut in the genome, resulting in a ~7 nt overhang.


Base Editor Cas9

Kinase dead and nickase form of Cas9 fused to cytidine deaminase specifically transitions C or G to T or A.

CRISPRa (CRISPR activation)

Activation of transcription by recruiting a kinase dead form of Cas9 fused to a VP16/VP64 protein to activate transcription.

CRISPRi (CRISPR interference)

Interference with transcription by recruiting a kinase dead form of Cas9, fused to a KRAB domain to approximately 150 bp up- or down stream of the transcription start site.