CRISPR makes it easy to target multiple loci - a concept called multiplexing. Since CRISPR is such a robust system, editing or labeling efficiency doesn’t usually change when you add multiple gRNAs. Sound good? Addgene has many tools to help you multiplex - we’ll use mammalian plasmids to introduce you to some of your potential options and cloning methods, but please scroll down for plasmids suitable for other model systems, including E. coli, plants, Drosophila, and zebrafish!
One common question Addgene Senior Scientists receive is: can I express more than one gRNA from a single promoter using a plasmid like pX330? Unfortunately, the short answer is no. Unless you use a system for processing a continuous multi-gRNA transcript, each gRNA must be expressed from its own promoter. But that doesn’t mean you have to clone and transfect multiple promoter-gRNA constructs in order to target multiple sites...
Let’s start with the simplest multiplexing situation: you only need to express two gRNAs at the same time. One system you could use is pX333 from the Ventura lab. pX333, a modification of pX330, contains humanized wtCas9 and two U6 promoters. To use this plasmid, you simply order oligonucleotides for your chosen gRNA target sequences and clone them in just as you would for a single gRNA. You’ll clone in the first gRNA using restriction enzyme BbsI and the second gRNA using restriction enzyme BsaI. If you’re working in Drosophila, a two-gRNA expressing plasmid is available from the Bullock lab, and gRNAs can be inserted using Gibson Assembly or SLIC cloning methods. A BsaI-based E. coli multiplexing plasmid is available from the Koffas lab.
Golden Gate Assembly Methods
If you want to scale up to 3-7 gRNAs, Addgene has two Golden Gate Assembly-based mammalian systems from the Gersbach and Yamamoto labs. Golden Gate assembly methods are also available for plants (Qi-Jun Chen and Yao-Guang Liu labs) and zebrafish (Wenbiao Chen lab).
Golden Gate Assembly uses Type IIS restriction enzymes, which cleave outside of their recognition sequence, creating flanking overhangs. These overhangs can be customized to link together multiple fragments, allowing ordered assembly of multiple components into a destination vector.
The first step in CRISPR/Cas9 Golden Gate multiplexing is to clone the oligonucleotides specifying each gRNA target sequence into distinct expression vectors using the enzyme BbsI. These expression vectors each contain Type IIS restriction sites flanking the promoter-gRNA construct, but with different sequences adjacent to the sites. When digested with the appropriate Type IIS enzyme, the unique flanking overhang sequences can link together to allow for ordered assembly into a destination vector that expresses Cas9. If you’re confused, check out the schematic below.
gRNA target sequences (colored rectangles) are cloned into various plasmids using oligonucleotides. These plasmids contain Type IIS restriction sites that flank the promoter-gRNA constructs. When these plasmids are digested, unique overhangs (here, O1-4) adjacent to the cut sites “link” fragments together and drive ordered assembly into a Cas9-containing destination vector. Note: depending on which method you use, the procedure will vary slightly.
This plasmid set allows you to express 2-4 gRNAs, with four being the ideal number. First you generate four unique kanamycin-resistant plasmids, each containing a different gRNA target sequence downstream of the 7SK, human U6, mouse U6, or human H1 promoters. If you express fewer than four gRNAs, you’ll clone in a polyT-termination sequence for each unused promoter. This step is necessary to generate all of the overhangs needed for the final ligation step. Plasmids are then digested using BsmBI and ligated into one of four Cas9 or dCas9-containing ampicillin-resistant destination vectors. Destination vector options include humanized wt Cas9, dCas9 (transcriptional repressor), and dCas9-VP64 (transcriptional activator)-containing plasmids. If you’re interested in combining CRISPR/Cas9 with FACS-based screening, these plasmids are for you. Each destination vector contains GFP, enabling you to select cells with high GFP expression and therefore high multiplicity of infection (MOI). These high MOI cells have the highest levels of Cas9 and gRNA expression, and thus the highest frequency of genome editing events.
This kit is built for serious multiplexing and enables users to express up to 7 gRNAs! The kit contains different destination vectors depending on the total number of gRNAs you wish to clone, from 2-7. For example, if you’re expressing 4 gRNAs, you’d use pX330A-1x4; for 6 gRNAs, you’d use pX330A-1x6. This customization means you don’t ever need to clone in filler sequences like with the Gersbach plasmids; the correct flanking regions are specified by the destination vector. To build your multiplexing construct, you clone all but one of your gRNAs into spectinomycin-resistant plasmids pX330S-2 to pX330S-(last gRNA number). The 5’ most gRNA is cloned into the Cas9-containing, ampillicin-resistant destination vector. These constructs are digested using BsaI and assembled to produce a plasmid encoding the gRNAs, Cas9, and ampicillin resistance. As with the Gersbach lab plasmids, multiple Cas9 variants are available. The standard kit includes wt humanized Cas9 and the D10A nickase mutant (Cas9n). The accessory pack adds two more options: dCas9 (transcriptional repression) and Fok1-dCas9 (dimeric nuclease).
Gateway Assembly Method
This kit can be used to create lentiviral vectors expressing wt humanized Cas9 and up to three gRNAs. Entry vectors containing the U6 promoter and the gRNA scaffold are provided with the kit. Oligonucleotides specifying the gRNA seed sequence should be compatible with type IIS enzyme BfuAI. Gateway cloning is then used to combine the multiple gRNAs and Cas9 together into a single plasmid. Although only wt hCas9 entry vectors are supplied with the kit, you can clone your own entry vectors containing other Cas9 variants to use with the MuLE system.
Multiplexing from a Single Transcript
You can also multiplex gRNAs via a polycistronic transcript. Rather than being transcribed from different promoters, the gRNAs are transcribed together and are flanked by specific sites that allow them to be cleaved and released. These constructs tend to be smaller than constructs with multiple promoter-gRNA cassettes, making them advantageous for small capacity vectors like AAV. In addition to the mammalian option described below, plasmids for making polycistronic gRNAs are also available from the Yang lab for use in plants.
The mammalian multiplex systems use the Csy4 RNA nuclease from Pseudomonas
aeruginosa. When overexpressed, Csy4 efficiently cleaves gRNAs sandwiched between 28 base Csy4 recognition sites. If Csy4 is not expressed, the gRNAs cannot be released, adding temporal and/or spatial control to the system. pSQT1313 from the Joung lab allows you to express two gRNAs constructed using oligonucleotide assembly. Unlike some of the plasmids described above, this vector do not contain Cas9, so you’ll need to supply it with another plasmid.
Multiplexing in PlantsQi-Jun Chen Lab Golden Gate/Gibson Assembly Multiplexing Plasmids:
These plasmids allow you to assemble 2-4 gRNAs through Golden Gate or Gibson Assembly. gRNAs are inserted into the pCBC vectors using BsaI, and promoter-gRNA fragments are PCR amplified for cloning into one of three Zeamays codon-optimized Cas9-containing binary vectors. These vectors are based off of pGreen, pSoup, and pCAMBIA, three of the most popular plant vectors that together are suitable for a wide variety of applications. The Chen lab system is compatible with both monocot and dicot plants.
These plasmids can be used to successfully express up to 8 gRNAs after Golden Gate or Gibson Assembly! Using BsaI, gRNAs are cloned into one of 12 pYLsGRNA plasmids, which contain various promoters and reporters, and subsequently inserted into a Cas9-containing destination vector based off of pCAMBIA. wt Cas9 is plant-optimized with a high 5’ GC-content, and plasmids are available for both monocot and dicot plants, with a choice of either hygromycin or Basta selection.
These plasmids are similar to the Csy4 polycistronic system described above, except that they use an endogenous nuclease system to cleave the gRNAs. gRNAs are flanked by glycine tRNAs to create polycistronic glycine tRNA-gRNA (PTG) constructs. Eukaryotic RNases P and Z recognize the tRNA sequences, cleave them, and release the gRNAs. PTGs are assembled into a wt Cas9-containing vector using Golden Gate assembly, and up to 8 gRNAs may be expressed simultaneously. Vectors for both transient expression and Agrobacterium-mediated transformation are available. For more information on this system, check out this blog post.
Multiplexing in Zebrafish
These plasmids allow expression of 2-5 gRNAs in zebrafish. As in the Yamamoto system, custom destination vectors are used depending upon the total number of gRNAs you wish to clone, so you don’t have to clone any filler sequences. You also have the option of including a previously-designed tyr gRNA, which causes hypopigmentation, thus marking cells that have undergone genomic modification. In this system, Cas9 must be supplied on a separate plasmid.
Multiplexing in Drosophila
Two gRNAs can be assembled using Gibson Assembly or SLIC cloning. gRNAs are expressed from two Drosophila U6 promoters. Cas9 must be supplied on a separate plasmid.
Multiplexing in E. coli
This system allows you to assemble type II-A CRISPR arrays for dCas9-based transcriptional repression. The CRISPathBrick plasmid contains a nontargeting spacer flanked by two CRISPR repeats. The spacer can be digesting using BsaI, allowing a spacer-repeat “brick” to be inserted. The BsaI site remains intact, allowing subsequent “bricks” to be added one by one. This approach is especially useful for combinatorial analyses. For example, if you were to develop an array using 3 distinct spacer-repeats (more are possible), you could easily create 7 unique arrays (e.g. for spacers A, B, and C, you could obtain arrays A, B, C, AB, AC, BC, and ABC).
Check out a curated, up-to-date list of our gRNA multiplexing vectors on our gRNA page!
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Additional Resources on the Addgene Blog
- Browse All of Our CRISPR 101 Posts
- Get Advice on Choosing The Proper Cas9 Variant for Your Experiment
- Learn How to Design Your gRNA
Additional Resources on Addgene.org
- Catch Up on Your CRISPR Background with Our Guide Pages
- Find CRISPR Plasmids for Your Research
- Browse Our CRISPR Multiplexing Resources