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NT-CRISPR Plasmid Collection
(Kit # 1000000189 )

Depositing Lab:   Anke Becker

The NT-CRISPR plasmid collection contains all plasmids to perform genome engineering in Vibrio natriegens using the NT-CRISPR method.

This kit will be sent as individual bacterial stabs.

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$400 USD + shipping

Available to academics and nonprofits only.

Original Publication

NT-CRISPR, combining natural transformation and CRISPR-Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens. Stukenberg D, Hoff J, Faber A, Becker A. Commun Biol. 2022 Mar 25;5(1):265; doi: 10.1038/s42003-022-03150-0 (Link opens in a new window) PMID: 35338236 (Link opens in a new window).

Description

The included plasmids can be used as follows:

  • pST_116_LVL2 cam: NT-CRISPR with a single gRNA using the regular S. pyogenes Cas9 (PAM = NGG).
  • pST_140_LVL2 cam: NT-CRISPR with a single gRNA using the spG Cas9, which allows alternative PAM sequences (NGT, NGC, NGA).
  • pST_119_LVL2 cam: Backbone plasmid for the assembly of NT-CRISPR plasmids with two to five gRNAs.
  • pMC0x_gRNA_Pos_Ptet: Different position vectors for gRNAs. gRNAs are first integrated in position vectors, which can then be used for the assembly of multi gRNA plasmids using pST_140_LVL2.
Schematic for inserting gRNAs into NT-CRISPR plasmids
  • Assembly scheme for NT-CRISPR plasmids with multiple gRNAs. gRNA spacer sequences are first introduced into different gRNA position vectors and are then combined with the pST_240_LVL2 plasmid to generate NT-CRISPR plasmids with multiple gRNAs. pST_240_LVL2 carries a sfGFP dropout part, which is replaced with the respective gRNA expression cassettes. Thereby, correct assembly is indicated by loss of the sfGFP signal yielding white instead of green colonies after transformation. Different gRNA position vectors are used, depending on the number of desired gRNAs in the final construct (Modified from Stukenberg et al. 2022 under CC-BY license (Link opens in a new window)).

How to Cite this Kit

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.

For your Materials and Methods section:

“The NT-CRISPR Plasmid Collection was a gift from Anke Becker (Addgene kit #1000000189).”

For your Reference section:

NT-CRISPR, combining natural transformation and CRISPR-Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens. Stukenberg D, Hoff J, Faber A, Becker A. Commun Biol. 2022 Mar 25;5(1):265; doi: 10.1038/s42003-022-03150-0 (Link opens in a new window) PMID: 35338236 (Link opens in a new window).

The NT-CRISPR Plasmid Collection contains 11 plasmids. Please refer to the individual plasmid pages below for more details on each plasmid in this kit:

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