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CRISPR-Cas9 Gateway System for Physcomitrella patens
(Kit # 1000000151 )

Depositing Lab:   Magdalena Bezanilla

The CRISPR-Cas9 Gateway System is a collection of 24 plasmids providing a modular, efficient CRISPR/Cas9 editing system for accurate and customizable repair in Physcomitrella patens.

This kit will be sent as bacterial glycerol stocks in 96-well plate format.

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$330 USD + shipping

Available to academics and nonprofits only.

Description

This collection of 24 plasmids provides a modular, efficient CRISPR/Cas9 editing system for accurate and customizable repair in Physcomitrella patens. Our vector system uses a Type IIS restriction enzyme to linearize entry vectors containing the sgRNA. Customized protospacer oligonucleotides can be easily, directionally, and seamlessly annealed into the entry clone. A final expression vector can be generated with one of three destination vectors that only differ in antibiotic resistance genes for selection in moss. The destination vectors express the Cas9 nuclease and the sgRNA. Multisite Gateway allows up to four entry vectors to recombine with a single destination vector. Co-transformation into protoplasts with all three destination vectors enables the ability to target up to 12 genomic sites.

Endogenous homology-directed repair can be used to insert DNA sequences at specific genomic sites. This is accomplished by co-transformation of the Cas9/sgRNA expression plasmid and a plasmid containing regions of homology surrounding the expected sgRNA cleavage site. Vectors for inserting sequences encoding fluorescent fusion proteins are provided. We have also included a Stop Codon Cassette, which contains stop codons in each frame ensuring the introduction of a stop codon very near the sgRNA cleavage site, as well as a multiple cloning site that aids with downstream genotyping.

bezanilla-crispr-schematic.png
Figure legend (A) A detailed plasmid map of pENTR-PpU6P-sgRNA (the entry vector). The P. patens U6 promoter and DNA encoding for an sgRNA are situated between MultiSite Gateway recombination (att) sites. Two inverted BsaI restriction sites exist between the U6 promoter and the sgRNA for protospacer ligation (red box). There are 7 plasmids available for each of the MultiSite Gateway entry vector att site combinations, listed in the box. (B) A simplified plasmid map of an entry vector showing the DNA sequence between the U6 promoter and the sgRNA. Linearization of the plasmid with BsaI results in unique ssDNA overhangs. Double-stranded oligonucleotide DNA containing the complementary overhangs and the gene-specific protospacer sequence are annealed into the linearized entry vector using a sticky-end ligase. (C) A detailed plasmid map of the various destination vectors. These plasmids contain the Cas9 coding sequence driven by the Maize ubiquitin promoter for constitutive expression of Cas9 in protoplasts, the Gateway cassette, and antibiotic resistance genes for selection in plants. Vectors included are pMH-Cas9-gate, pMK-Cas9-gate, and pZeo-Cas9-gate for selection on hygromycin, G418, and Zeocin, respectively. (D) An illustration showing a simple Gateway LR Reaction involving att site recombination of a gene-specific entry vector (top) and a destination vector (center) to yield a Cas9/sgRNA expression vector (bottom). Sequence features are not drawn to scale. (E) Illustrations showing the MultiSite Gateway LR Reactions. Multiple entry vectors can undergo recombination with a single destination vector. Up to four entry vectors can be recombined into a single destination vector to yield a vector that expresses four sgRNAs and the Cas9 nuclease. Sequence features are not drawn to scale. (F) An example of accurate repair using the endogenous homology-directed repair mechanisms to insert a gene tag. Recombination of a three element Gateway LR reaction (top left) involving entry clones containing regions of homology surrounding the expected Cas9 cleavage site (blue and yellow) and a GFP (green) with a destination vector, pGEM-gate, to yield pGEM-GFP-HDR (bottom left). Co-transformation of pGEM-GFP-HDR with the Cas9/sgRNA expression plasmid leads to accurate DNA repair with an in-frame GFP (right). (G) A simplified plasmid map of a Stop Codon Cassette vector, an entry vector containing stop codons in all three frames near the 5’ end (top left). A MultiSite LR reaction with entry vectors containing upstream and downstream sequences surrounding the expected Cas9 cleavage site (blue and yellow, respectively) and the stop codon cassette entry vector recombine with pGEM-gate. This yields a plasmid, pGEM-stop-HDR, that can be co-transformed with the Cas9/sgRNA expression plasmid for accurate integration of early stop codons into your gene-of-interest.

Kit Documentation

Icon Bezanilla Lab - CRISPER-Cas9 + HDR protocol (133.4 KB)

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 CRISPR-Cas9 Gateway System for Physcomitrella patens was a gift from Magdalena Bezanilla (Addgene kit #1000000151).”

CRISPR-Cas9 Gateway System for Physcomitrella patens - #1000000151

Resistance Color Key

Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.

Inventory

Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.

Kit Plate Map

96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.

Resistance Color Key

Kanamycin
Chloramphenicol and Ampicillin

Inventory

Well Plasmid Resistance
A / 1 pENTR-PpU6P-sgRNA-L1L2
Kanamycin
A / 2 pENTR-PpU6P-sgRNA-L1L4
Kanamycin
A / 3 pENTR-PpU6P-sgRNA-L1R5
Kanamycin
A / 4 pENTR-PpU6P-sgRNA-L5L2
Kanamycin
A / 5 pENTR-PpU6P-sgRNA-R4R3
Kanamycin
A / 6 pENTR-PpU6P-sgRNA-L3L2
Kanamycin
A / 7 pENTR-PpU6P-sgRNA-L5L4
Kanamycin
A / 8 pENTR-R4R3-stop
Kanamycin
A / 9 pENTR-R4R3-mEGFP-N
Kanamycin
A / 10 pENTR-R4R3-2xmEGFP-N
Kanamycin
A / 11 pENTR-R4R3-3xmEGFP-N
Kanamycin
A / 12 pENTR-R4R3-mEGFP-C
Kanamycin
B / 1 pENTR-R4R3-2xmEGFP-C
Kanamycin
B / 2 pENTR-R4R3-3xmEGFP-C
Kanamycin
B / 3 pENTR-R4R3-2xmRuby-N
Kanamycin
B / 4 pENTR-R4R3-3xmRuby-N
Kanamycin
B / 5 pENTR-R4R3-mRuby-C
Kanamycin
B / 6 pENTR-R4R3-2xmRuby-C
Kanamycin
B / 7 pENTR-R4R3-3xmRuby-C
Kanamycin
B / 8 pMH-Cas9-gate
Chloramphenicol and Ampicillin
B / 9 pMK-Cas9-gate
Chloramphenicol and Ampicillin
B / 10 pGEM-gate
Chloramphenicol and Ampicillin
B / 11 pZeo-Cas9-gate
Chloramphenicol and Ampicillin
B / 12 pENTR-R4R3-mRuby-N
Kanamycin
C / 1 IG703: pMVP (L1-R5) TRE promoter
Kanamycin
Data calculated @ 2019-07-18

Kit Plate Map - #1000000151

Plate #1

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