I am using CRISPR Cas9 system to knock out my gene of interest in Nicotiana tobacum. For this I need a complete protocol explaining each and every step with reaction mixture. Anybody sharing a complete protocol will be very helpful to me.
Thank you
1. Please refer to Addgene (CRISPR) website [see links below]. They have pretty detailed description of this technology (including their components) and the details of designing plasmids (almost step-by-step). It will be very useful in designing and cloning your vectors.
(a) 'CRISPR/Cas9 Guide' https://www.addgene.org/crispr/guide/#plan-experiment
(b) 'Plan Your CRISPR Experiment' https://www.addgene.org/crispr/guide/#plan-experiment
(c) 'CRISPR protocols' (In this page, you can see some detailed protocols (downloadable PDF) from different research labs. You can use them as references. https://www.addgene.org/crispr/reference/#protocols
(d) 'CRISPR/Cas9 Plasmids for use in Plants' https://www.addgene.org/crispr/plant/
2. See the attached paper (published in Plant Methods). This is a review paper describing the CRISPR system (components, including different promoters) used in plants by 2013.
Paper title: "Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system".
you could also see the following in Nat Biotechnol (2013)
doi: 10.1038/nbt.2654
Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9
good luck.
@Saroj,
Attached is an 2016 book chapter from Methods in Molecular Biology: Chromosome and Genomic Engineering in Plants-Methods and Protocols. In this chapter, the authors describe the detailed protocol of using CRISPR/Cas9 in Arabidopsis mutagenesis. It says the protocol can also apply to other plant species. This is particular useful because it includes all the steps, chemicals and detergents.
The article is obtained from this web page: CRISPR/Cas Plasmids & Protocols for Genome Engineering in Plants. http://www.botanik.kit.edu/molbio/940.php
The author professor Dr. Puchta Holger is one of the pioneers in studying gene targeting. He has quite a few reviews focusing on 'gene-targeting'.
Thank you for the information......
While going through the many papers related to the CRISPR protocol following I found very useful and like to share to other who are looking for the similar protocol
@ Saroj,
Thanks for the attachments.
The third attachment 'LentiCRISPR' is indented for mammalian cells, such as human being. It should be delivered by lentivirus. The CRISPR/Cas9 components on the plasmid, including the promoters and selection markers (and probably others), are optimized for mammalian cells, not for plants. But, the detailed description of designing the sgRNA in section 'Target Guide Sequence Cloning Protocol' should be very useful.
I think this protocol is from Dr. Feng Zhang's lab at MIT. Now he is a big shot (one of the creators of CRISPR/Cas9 technology) because of this technology. His institute/lab is fighting the CRISPR patents with UC-Berkeley Dr. Jennifer Doudna's group right now.
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