CRISPR Plasmids - Protein Tagging
The CRISPR system has been adapted to allow tagging of proteins expressed from their natural chromosomal context. These CRISPR tagging methods allow for improved efficiency and throughput over traditional tagging methods. Several different tagging techniques, as well as the plasmids and protocols needed for using them, are described below.
Read our CRISPR 101 post for more information about HDR/repair template design.
Mendenhall and Myers Tagging System
The Eric Mendenhall and Richard Myers labs have deposited plasmids for a CRISPR-based system to add a tag (currently using FLAG) to endogenous proteins. This CRISPR/Cas plasmid-system consists of two components:
- A vector containing Cas9 and a validated gRNA, based on the Zhang lab's PX458.
- Multiple gRNA plasmids may be used.
- The HDR (homology directed repair) donor plasmid with homology arms and selection marker.
The first deposited plasmids in this CRISPR-Cas tagging system were tested by tagging transcription factors with FLAG in human cell lines. To repeat the tagging in your system, use the plasmids as listed in each row. If your own gene of interest is currently unavailable, you will need to design and clone in a gRNA(s) to guide the Cas9 protein to your target sequence, as well as design and clone in the homology arms for the donor plasmid.
The Mendenhall and Myers labs have also provided their protocol for homology arm cloning:Mendenhall & Myers FETCh-seq Protocol 134.7 KB
Mendenhall and Myers Tagging Plasmids
Doyon Tagging System
Recently, Yannick Doyon's lab deposited a plasmid which introduces an N- or C- terminal affinity tag (3xFLAG-2xSTREP) on endogenous genes for the isolation of native protein complexes. This vector serves as a backbone to clone the left and right homology arms. More details on how to use this plasmid in your experiments can be found below and in the supplemental materials from the Doyon Lab’s publication: Dalvai et al. Cell Rep. 2015. Alternatively, cDNAs can be cloned directly into this vector and targeted to the AAVS1 genomic safe harbor locus using untagged SpCas9 (Addgene 41815 or #44719) in combination with gRNA_AAVS1-T2 (Addgene #41818) or using an all in one vector from the Doyon lab, eSpCas9(1.1)_No_FLAG_AAVS1_T2 (Addgene #79888), which expresses an untagged Cas9 AND a gRNA targeting the AAVS1 locus.
- Doyon Tagging Plasmid: 3xFLAG-2xSTREP
- Construct for integrating at the AAVS1 "safe harbor" locus: eSpCas9(1.1)_No_FLAG_AAVS1_T2
Yamamoto PITCh Tagging System
The Takashi Yamamoto lab has deposited the PITCh system plasmids for CRISPR-based knock-in of EGFP-2A-PuroR cassette to the C-terminus of endogenous proteins. The PITCh system depends on MMEJ (microhomology-mediated end-joining), an alternative DSB repair pathway, instead of HDR, which makes it easy to construct a donor vector, because MMEJ utilizes very short (around 20 bp) microhomologies as homology arms. The system consists of two components:
- An all-in-one CRISPR/Cas9 vector based on the Zhang lab's PX330, simultaneously targeting the genomic site and the donor vector.
- The PITCh donor plasmid with an EGFP-2A-PuroR cassette, flanked by microhomologous sequences and gRNA target sites.
The first deposited PITCh plasmids were tested by fusing EGFP-2A-PuroR cassette to a nucleolar protein, fibrillarin (FBL). To repeat the knock-in in your system, use the plasmids as listed below. When targeting other gene loci, you will prepare the gene-specific CRISPR and donor vectors as well as pX330S-2-PITCh, expressing a generic gRNA targeting the PITCh donor plasmid. Note that the principle of the PITCh system is different from the conventional HDR-mediated gene knock-in. The detailed procedure can be found in Sakuma et al., Nature Protocols, 2016.
- Yamamoto Tagging Plasmids:
- pX330S-2-PITCh- expresses Cas9 & PITCh-gRNA
- pX330A-FBL/PITCh- expresses Cas9, PITCh-gRNA, & FBL-specific gRNA
- pCRIS-PITChv2-FBL- PITCh donor vector for EGFP-2A-PuroR knock-in into human FBL locus
Jorgensen Lab SapTrap CRISPR/Cas Toolkit
SapTrap is a modular toolkit from Erik Jorgensen's lab for building CRISPR targeting vectors to insert genetic tags in the C. elegans genome. The SapTrap reaction produces a single plasmid targeting vector that encodes both a guide RNA transcript and a repair template for an individual tagging event. The kit contains 26 plasmids; 21 of the plasmids are for use in SapTrap reactions for targeting vector assembly, and the remaining 5 plasmids include Cre and FLP expression vectors, a general cloning plasmid, and a prebuilt Unc-32::GFP targeting vector.
Kanemaki Lab Auxin-Inducible Degron Tagging
Masato Kanemaki's lab has developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins by using donor constructs that harbor synthetic short homology arms. Notably, they have used Auxin-inducible degron (AID) tagging with CRISPR/Cas to generate conditional alleles of essential nuclear and cytoplasmic proteins, which can then be depleted very rapidly after the addition of auxin to the culture medium. Plasmids can be found associated with the following article:
Förstemann Drosophila Cell Tagging System
The Förstemann lab has developed a CRISPR tagging technique for use in Drosophila cells that uses PCR to generate both an expression cassette for the Cas9-programming sgRNA and HR donors for selectable genome modification. This system can be used to create C- and N-terminal epitope tags. The plasmids in the following articles provide PCR templates for amplification of the tag (eg GFP, Flag, YFP, etc) and selection markers. Two independent selection markers are available. An improved FLP recombinase expression vector allows for efficient marker cassette FLP-out. Plasmids can be found associated with the following two articles:
The Förstemann lab has provided detailed protocols for N- or C-terminal tagging in Drosophila cells.N terminal tagging in Drosophila cells 3.2 MB
C terminal tagging in Drosophila cells 3.3 MB
Seydoux C. elegans Tagging System
Geraldine Seydoux's lab has developed a systematic and scalable method to create marker-free mutations, insertions, and deletions at any locus in C. elegans . This system uses a 10-day protocol, generates “clean” homozygous mutants with no co-integrated markers or footprints, and can be scaled up for systematic editing of multiple genes. Plasmids can be found associated with the following article:
Allen Institute for Cell Science Plasmid Collection
The Allen Institute for Cell Science has produced the first publicly available collection of fluorescently tagged, human, induced pluripotent stem cell (hiPSC) lines. These cell lines were created using CRISPR and the donor plasmids containing homology arms and EGFP are available at Addgene.
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