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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.

How to use CRISPR to tag your gene of interest

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

Protein Species Tag Donor Plasmid gRNA plasmid gRNA plasmid
FLAG pFETCh_Donor
CEBPB Human FLAG pFETCh_CEBPB PX458_CEBPB_1 PX458_CEBPB_2
AHR Human FLAG pFETCh_AHR PX458_AHR_1 PX458_AHR_2
ARID4B Human FLAG pFETCh_ARID4B PX458_ARID4B_1
BCL6_iso1 Human FLAG pFETCh_BCL6_iso1 PX458_BCL6_iso1_1 PX458_BCL6_iso1_2
CEBPG Human FLAG pFETCh_CEBPG PX458_CEBPG_1 PX458_CEBPG_2
RAD21 Human FLAG pFETCh_RAD21 PX458_RAD21
GABPA Human FLAG pFETCh_GABPA PX458_GABPA_1 PX458_GABPA_2
GABPB1_iso2 Human FLAG pFETCh_GABPB1_iso2 PX458_GABPB1_iso2_1
ATF1 Human FLAG pFETCh_ATF1 PX458_ATF1
CEBPA Human FLAG pFETCh_CEBPA PX458_CEBPA_1 PX458_CEBPA_2
CREB1 Human FLAG pFETCh_CREB1 PX458_CREB_1 PX458_CREB_2
TGIF2 Human FLAG pFETCh_TGIF2 PX458_TGIF2_1 PX458_TGIF2_2
SSRP1 Human FLAG pFETCh_SSRP1 PX458_SSRP1_1 PX458_SSRP1_2
ZNF146 Human FLAG pFETCh_ZNF146 PX458_ZNF146_1 PX458_ZNF146_2
ZNF580 Human FLAG pFETCh_ZNF580 PX458_ZNF580_1 PX458_ZNF580_2
DNMT3B Human FLAG pFETCh_DNMT3B PX458_DNMT3B
ZNF219 Human FLAG pFETCh_ZNF219 PX458_ZNF219_1 PX458_ZNF219_2
ZNF3 Human FLAG pFETCh_ZNF3 PX458_ZNF3_1 PX458_ZNF3_2
FOXP1 Human FLAG pFETCh_FOXP1 PX458_FOXP1_1 PX458_FOXP1_2
GATAD1 Human FLAG pFETCh_GATAD1 PX458_GATAD1_1 PX458_GATAD1_2
GATAD2A Human FLAG pFETCh_GATAD2A PX458_GATAD2A_1 PX458_GATAD2A_2
HHEX Human FLAG pFETCh_HHEX PX458_HHEX_1 PX458_HHEX_2
HBP1 Human FLAG pFETCh_HBP1 PX458_HBP1_1
HLF1 Human FLAG pFETCh_HLF PX458_HLF_1 PX458_HLF_2
HMG20A Human FLAG pFETCh_HMG20A PX458_HMG20A_1 PX458_HMG20A_2
HOMEZ Human FLAG pFETCh_HOMEZ PX458_HOMEZ_1 PX458_HOMEZ_2
KAT7 Human FLAG pFETCh_KAT7 PX458_KAT7_1
KDM6A Human FLAG pFETCh_KDM6A PX458_KDM6A_1 PX458_KDM6A_2
KLF9 Human FLAG pFETCh_KLF9 PX458_KLF9_1 PX458_KLF9_2
KLF11 Human FLAG pFETCh_KLF11 PX458_KLF11_1 PX458_KLF11_2
KMT2B Human FLAG pFETCh_KMT2B PX458_KMT2B_1 PX458_KMT2B_2
MBD1_iso1 Human FLAG pFETCh_MBD1_iso1 PX458_MBD1_iso1_1 PX458_MBD1_iso1_2
MIER3 Human FLAG pFETCh_MIER3 PX458_MIER3_1 PX458_MIER3_2
MIXL1 Human FLAG pFETCh_MIXL1 PX458_MIXL1_1 PX458_MIXL1_2
MLX Human FLAG pFETCh_MLX PX458_MLX_1 PX458_MLX_2
NCoA2 Human FLAG pFETCh_NCoA2 PX458_NCoA2_1
NFIA Human FLAG pFETCh_NFIA_iso1 PX458_NFIA_iso1_1 PX458_NFIA_iso1_2
NFIL3 Human FLAG pFETCh_NFIL3 PX458_NFIL3_1 PX458_NFIL3_2
NR2F6 Human FLAG pFETCh_NR2F6 PX458_NR2F6_1 PX458_NR2F6_2
PBX2 Human FLAG pFETCh_PBX2 PX458_PBX2_1 PX458_PBX2_2
RERE Human FLAG pFETCh_RERE PX458_RERE_1
RFXANK Human FLAG pFETCh_RFXANK PX458_RFXANK_1 PX458_RFXANK_2
RXRB Human FLAG pFETCh_RXRB PX458_RXRB_1 PX458_RXRB_2
SAP130 Human FLAG pFETCh_SAP130 PX458_SAP130_1 PX458_SAP130_2
SOX5 Human FLAG pFETCh_SOX5 PX458_SOX5_1
SP5 Human FLAG pFETCh_SP5 PX458_SP5_1 PX458_SP5_2
TEAD1 Human FLAG pFETCh_TEAD1 PX458_TEAD1_1
TEAD3 Human FLAG pFETCh_TEAD3 PX458_TEAD3_1 PX458_TEAD3_2
ZFP1_iso1 Human FLAG pFETCh_ZFP1_iso1 PX458_ZFP1_iso1_1 PX458_ZFP1_iso1_2
ZGPAT Human FLAG pFETCh_ZGPAT PX458_ZGPAT_1 PX458_ZGPAT_2
ZNF644 Human FLAG pFETCh_ZNF644 PX458_ZNF644_1 PX458_ZNF644_2
ZNF652 Human FLAG pFETCh_ZNF652 PX458_ZNF652_1 PX458_ZNF652_2
ZNF792 Human FLAG pFETCh_ZNF792 PX458_ZNF792_1 PX458_ZNF792_2
ZSCAN9 Human FLAG pFETCh_ZSCAN9 PX458_ZSCAN9_1 PX458_ZSCAN9_2
ATF4 Human FLAG pFETCh_ATF4 PX458_ATF4_1 PX458_ATF4_2
ARID5B Human FLAG pFETCh_ARID5B PX458_ARID5B_1 PX458_ARID5B_2

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 Lab TAP Tagging Protocol 97.2 KB

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.

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.


Do you have suggestions for other plasmids that should be added to this list?

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