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.

Original Publication

Efficient and modular CRISPR-Cas9 vector system for Physcomitrella patens. Mallett DR, Chang M, Cheng X, Bezanilla M. Plant Direct. 2019 Sep 12;3(9):e00168. doi: 10.1002/pld3.168. PubMed PMID: 31523744.

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.

QC Note: Analysis of pENTR-R4R3-stop shows that the stop cassette has a tendancy to dimerize, which may result in diagnostic restriction digests or Sanger sequencing results that are inconsistent with the full plasmid sequence. The depositing laboratory has confirmed that this should not affect plasmid function.

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.
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Kit Documentation

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).”

For your Reference section:

Efficient and modular CRISPR-Cas9 vector system for Physcomitrella patens. Mallett DR, Chang M, Cheng X, Bezanilla M. Plant Direct. 2019 Sep 12;3(9):e00168. doi: 10.1002/pld3.168. PubMed PMID: 31523744.

CRISPR-Cas9 Gateway System for Physcomitrella patens - #1000000151

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

Data calculated @ 2024-04-16