CRISPR Plasmids: Empty gRNA Expression Vectors
CRISPR requires that you have to express both a Cas protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the gRNA and a Cas protein act as an all-in-one vector, but their function (cut, nick, activate, interfere, etc.) is limited to that of the Cas protein present on the plasmid. gRNA plasmids that do not co-express a Cas protein require a separate plasmid that does so; however, these independent gRNA plasmids can be paired with a wide variety of plasmids and therefore are not limited to a single CRISPR function. Alternatively, gRNA-only plasmids can be used in cells or organisms in which Cas9 has been integrated into the genome.
gRNA Empty Vectors
Many gRNA empty vectors have been deposited at Addgene. To find the gRNA vector for your system, you can search or sort this table based on:
- Your particular expression system, eg. Mammalian, Lentiviral, AAV, Bacteria, Drosophila, Yeast, Plant, Xenopus, Worm, or Other.
- Whether the plasmid contains Cas9 and if so, which function of Cas9. We have plasmids with Cas9 that can cut, activate, interfere, or nick.
- Selection, such as Puromycin or EGFP
- Cloning enzyme used for insertion of your gRNA sequence, such as BsbI or BsaI
- Cas9 (or other CRISPR) system that the vector was designed to be used with, such as S. pyogenes, S. aureus, N. meningitidis, etc. gRNA scaffolds are specific for the species of Cas9 it is to be used with. crRNAs used with Cpf1 or other CRISPR systems are also listed here and denoted as such.
Want more information on the wide variety of Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme activity, experimental considerations, and more, created and maintained by the Doudna Lab.
Multiplex gRNA Vectors
Several systems have been developed to generate multiplex gRNA vectors. These systems can be used to express multiple gRNAs from a single plasmid with or without expression of Cas9. Highlighted below are some multiplex gRNA vectors from our collection.
| Plasmids | Expression System | Description | PI |
|---|---|---|---|
| Multiplex CRISPR/Cas9 Assembly System Kit | Mammalian | A system for constructing all-in-one expression vectors containing multiple guide RNA expression cassettes and a Cas9 nuclease/nickase expression cassette using the Golden Gate cloning method. A separate accessory pack is available for dCas9 and FokI-dCas9. | Yamamoto |
| Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector | Mammalian | Lentiviral expression of Cas9, dCas9, or dCas9-VP64 along with 1-4 sgRNAs expressed from independent promoters. sgRNAs are cloned into separate vectors and then assembled into one construct using Golden Gate assembly. | Gersbach |
| MuLE System | Mammalian | MultiSite Gateway recombination-based cloning of complex polycistronic lentiviruses. Can express Cas9 with up to 3 gRNAs. | Frew |
| pSQT1313 | Mammalian | Single plasmid for the expression of multipe gRNAs. Csy4 cleavable cassettes; requires expression of Csy4 (eg Plasmid 53369). | Joung |
| pX333 | Mammalian | Single all-in-one plasmid for tandem expression of two sgRNAs from independent U6 promoters and Cas9 from CBh promoter. | Ventura |
| A CRISPR/Cas9 toolkit for multiplex genome editing in plants | Plant | Plasmids for six gRNA module vectors, including three designed for dicots and three designed for monocots. Using these gRNA module vectors, multiple gRNA expression cassettes can be assembled using Golden Gate cloning or the Gibson Assembly. | Chen |
| Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system | Plant | Utilizes the tRNA processing system to reduce size and allow for expression of more sgRNAs in a single vector. sgRNAs are expressed as polycistronic glycine tRNA-gRNA genes (PTGs) and then PTG cassettes are assembled into a Cas9 expressing vector. | Yang |
| A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants | Plant | PCR-based procedure to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly | Liu |
| A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation | Plant | Three types of plasmids available for use in this Golden Gate and Gateway based assembly method: plasmids for assembly of single gRNA vectors with various promoters, plasmids for assembling multiple gRNAs into one vector, and plasmids that provide Cas9 variants. All components are then assembled into a single T-DNA binary vector of choice through Gateway recombination. | Qi |
| Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs | Zebrafish | Plasmids for assembly of gRNA cassettes using Golden Gate ligation into Tol2-based pGGDestTol2LC vector. Uses five distinct zebrafish U6 promoters for sgRNA expression. | Chen |
| pCFD4-U6:1_U6:3tandemgRNAs | Drosophila | Single plasmid for the expression of two gRNAs from Drosophila U6:1 and U6:3 promoters. | Bullock |
| pCFD5 | Drosophila | Utilizes endogenous tRNA processing machinery to excise multiple functional gRNAs from a single precursor transcript | Bullock |
| pCRISPathBrick | Bacteria | Allows rapid modular assembly of natural type II-A CRISPR arrays capable of simultaneously repressing multiple target genes in Escherichia coli. | Koffas |
Do you have suggestions for other plasmids that should be added to this list?
Fill out our Suggest a Plasmid form or e-mail help@addgene.org to help us improve this resource!