If you’ve ever been looking for just the right CRISPR vectors on Addgene and found instead ones that were… pretty close, or at least close enough, you’ve found yourself with a common dilemma. Request the vectors you can find and use them as-is, saving time and effort but risking the potential of them not working, or spend a few weeks designing and cloning the vectors you actually want. But now you can use the recently developed third option: Fragmid.
Fragmid is a modular vector assembly process designed for a quick, plug-and-play approach to cloning CRISPR vectors (McGee et al., 2024). It reduces the amount of time to design and create a new vector to four days for an experienced bench scientist. New to the bench? Fragmid depositor John Doench reports even the greenest members of his lab, high school interns, were able to use Fragmid to design and clone their vectors in five days.
Did you say… easier?
Fragmid’s modular assembly structure takes a lot of the design work out of the plasmid creation process. Each module of Fragmid represents a class of components, such as your CRISPR enzyme or Pol 2 promoter. The options available in each class, or module, are the components. Using the handy website, users can simply select the components they want for each module, generate the vector file, and quickly clone their vector of interest.
And it truly is plug-and-play. Because of its modular cloning approach, it’s easy to switch out reporters, Cas proteins, guide cassettes, promoters, N’ and/or C’ terminus, and selection markers. Need fewer components? A spacer can be inserted instead, allowing everything to stay in the correct order. Most Fragmid components are available for request on the Addgene website, both as individual plasmids and as the Fragmid kit.
You can also browse all Fragmid components on the Fragmid website.
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| Figure 1: (Top) Schematic overview and timeline of the Golden Gate (GG) cloning approach. Individual modules and a sample ligation are depicted. (Bottom) Schematic depicting available destination vectors for various delivery methods, along with compatible inserts. All destination vectors contain an ampicillin resistance cassette and an origin of replication, which are not schematized. Sizes of fragments are not to scale. Figure and caption from McGee et al., 2024, used under the Creative Commons CC BY-NC-ND license. |
Once you have the components you want, simply assemble them into a destination vector via Golden Gate cloning, perform an exonuclease cleanup, transform, plate, and pick. In short, the Fragmid system takes the heavy lifting out of CRISPR vector design, making it easier to get the vectors that are just right for your experiment.
As always, we recommend checking your cloning via a miniprep and restriction digest, and then plasmid sequencing if you can. To quote Doench, “Whole plasmid sequencing is just part of doing good science!”
Cloning specifics
Fragmid uses Bbsl, a Type II restriction enzyme that cleaves outside of its recognition sequence. This means that each component can be designed with an overhang specific to the type of module it is (Pol 2 protomer, Cas protein, ect...). Whatever individual components you choose will assemble in the expected module order. (It’s also why you can’t skip a module entirely, but instead will have to replace it with a spacer.)
Each component available on the Fragmid website, or through Addgene, has worked well in the hands of the Doench lab. Additionally, controls available in the system are validated and were chosen because of their broad applicability in different model systems.
What’s the error rate?
The Doench lab tested the Fragmid system by sequencing 60 individual assemblies created over five months. When they tested those clones (in duplicate), 93% had the correct banding pattern in the restriction enzyme digest. They sent 82 of the correctly banded clones for plasmid sequencing and found 98% of the plasmids were perfectly correct. Because they created the assemblies in duplicate, they were able to confirm that at least one plasmid was the correct sequence for every individual assembly.
What destination vectors are available?
What you’re building is important — but so is where you’re building it! Table 1 has a list of Fragmid destination vectors currently available.
Table 1: Fragmid destination vectors
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Organism |
Delivery Type |
Destination Vector |
|
Mammalian |
Lentivirus |
pRDA_512 (RSV-driven; lentiGuide-based) |
|
Mammalian |
Lentivirus |
pRDA_722 (CMV-driven; lentiCRISPRv2-based; preferred over 512) |
|
Mammalian |
Lentivirus |
pRDA_789 (CMV-driven; CROPseq) |
|
Mammalian |
AAV |
pRDA_889 (ITRs from AAV2) |
|
Mammalian |
Transposon |
pRDA_757 (Piggybac; constitutive expression) |
|
Mammalian |
Plasmid |
pRDA_791 (contains only orio and ampR) |
|
Drosophilia |
Plasmid |
pRDB_051 (for stable fly cell lines; attL and attR sequences for genome integration by phiC31) |
|
Drosophilia |
Plasmid |
pRDB_052 (for stable fly cell lines; Hygromycin resistance gene; for genome integration by spontaneous insertion) |
|
Drosophilia |
Plasmid |
pRDB_053 (for transgenic flies; gypsy insulator sequences; white + selectable marker; attB site for genome integration by phiC31 |
The Fragmid website
While the modular cloning approach is key to the Fragmid system, the Fragmid website, which breaks the design process down into four easy steps, provides the real value for the user. Let's walk through designing a CRISPR vector the Fragmid way!
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| Figure 1: Selecting your vector type on the Fragmid website. |
In step one, you’ll select the features of your vector, which are:
- Organism (Mammalian or drosophila)
- Delivery type (lentivirus, AAV, transposon, Plasmid)
- Destination vector (dropdown options depending on above selections)
- Module set (the set of components you want in your vectors)
- Target type (drop down options depending on above selections.)
- CRISPR enzyme (Cas9 [sp], Cas9 [other], Cas12a, RNA-targeting)
- CRISPR mechanism (KO, CRISPRa, CRISPRi, editing)
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| Figure 2: Selecting module fragments on the Fragmid website. |
Step two will ask you to fill in module fragments from a dropdown list for every module. Both the modules and the components available will depend on your answers in step 1, so you’ll only be able to select components that work with your design. Note that you can browse all components by clicking the blue text in the upper right-hand corner.
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| Step 3: Reviewing the plasmid summary on the Fragmid website. |
Step 3 is simply a review of the assembled plasmid. It shows you each component, with the ID of the component plasmid, component name, size, and any comments. You can use the ID to look up the component plasmids on the Addgene website.
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| Step 4: Naming your assembled plasmid and generating the vector file on the Fragmid website. |
If the vector is to your liking, your last step, Step 4, is to give the assembled plasmid a name and generate the vector file. Ta-da!
Most (180/181) Fragmid components are available in the Fragmid kit. While you can order each of the plasmids individually, if you’re going to be using more than six or seven components — in most cases, that's making two or more plasmids — it may be more efficient to order the kit.
What if Fragmid doesn’t have the components I want?
You can make your own Fragmid components! Ensure your backbone and genes contain only the Bbsl cut sites needed for the Fragmid cuts, clone in the appropriate overhang for your module type, and voilà! You’ll have your own Fragmid component (which you can also deposit with Addgene!)
We’re excited to help the Doench lab share this new tool and hope it helps you create great plasmids for your next CRISPR experiment. Happy cloning!
References and Resources
References
McGee, A. V., Liu, Y. V., Griffith, A. L., Szegletes, Z. M., Wen, B., Kraus, C., Miller, N. W., Steger, R. J., Escude Velasco, B., Bosch, J. A., Zirin, J. D., Viswanatha, R., Sontheimer, E. J., Goodale, A., Greene, M. A., Green, T. M., & Doench, J. G. (2024). Modular vector assembly enables rapid assessment of emerging CRISPR technologies. Cell Genomics, 4(3), 100519. https://doi.org/10.1016/j.xgen.2024.100519
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