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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.
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.
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.