Three Ways Gibson Assembly Strengthens CRISPR Workflows

CRISPR-Cas systems are programmable endonucleases that, since their discovery, have rapidly reshaped the fields of genome editing and gene therapy. The Cas protein, in complex with a single guide RNA (sgRNA), can be directed to nearly any DNA sequence. This gives basic researchers and drug developers in almost every biological field the ability to knock out—through nucleotide insertion or deletion (indel) via non-homologous end-joining (NHEJ) DNA repair—or knock in—using donor DNA and cell-mediated homology-directed repair – nearly any gene (or genes) of interest [1,2].

Wielding this technology, either for preclinical R&D or therapeutics development, requires the ability to manipulate and test various Cas proteins, sgRNAs, and other genes common in molecular biology, including fluorescent or luminescent reporters, promoters of different strengths, transcriptional terminators, and more. Accomplishing this requires a modular approach to constructing recombinant DNA, a robust cloning workflow, and rapid access to custom PCR primers and ready-made vectors.

These requirements can act as a major bottleneck to implementing CRISPR protocols in several model systems. Commonly, especially when implementing CRISPR-Cas expression in mammalian systems using lentiviruses, researchers can face problems with unstable inserts that undergo homologous recombination during the cloning process. One way around these difficulties is by using a purified ribonucleoprotein (RNP) complex, containing a Cas protein and sgRNA, for genome editing. Briefly, RNPs are assembled in vitro and transfected or electroporated into the desired cells. While this methodology presents significant advantages (i.e., circumventing expression or off-target editing complications), some experimental questions may require the use of reporter or selection markers, which necessitates the use of plasmid or virus-based systems, constructed using recombinant DNA technology.

Reliance on conventional cloning workflows—which use restriction enzymes—can present additional hurdles for molecular biologists looking to harness the power of CRISPR-Cas systems in their research. Commonly used restriction enzymes cut inverted repeat palindromic hexanucleotide sequences and dependence on these restriction sites has limitations: They must be incorporated into DNA fragments before cloning which can introduce undesirable sequences (also called “scars”) into gene products. Restriction enzymes also have varying degrees of activity, which can lead to inefficiencies in DNA digestion. Finally, traditional restriction cloning of multiple fragments can be burdensome, requiring subsequent rounds of multi-day digestion and screening protocols to obtain the desired DNA construct.

While restriction enzyme-based cloning methods have their place in molecular biology, modern-day cloning workflows required for CRISPR research need to be streamlined and rapid.

In 2009, a new cloning method—called Gibson Assembly—changed the way molecular cloning was done, largely solving many of the problems posed by conventional restriction enzyme-based methods and enabling seamless cloning, without the need for introducing restriction sites [3].

The reaction is a one-pot, isothermal reaction that can stitch up to 15 DNA fragments—with short homology regions from 20 to 40 nucleotides—together in approximately an hour (Figure 1). While efficiency does drop with multiple fragments, success rates are fairly high and can be done in as little as three days. The technique is uniquely suited for the modular DNA shuffling required for CRISPR applications. Below, we review a few recent breakthrough applications of CRISPR/Cas, all of which are powered by Gibson Assembly.
 

Many GEMMs that mimic different clinical aspects of cancer have been developed, helping cancer researchers untangle the process of tumor evolution and metastasis. For instance, through conditional deletion of Trp53 and Rb1 in the lung epithelium, researchers have developed a robust mouse model for small cell lung cancer (mSCLC) [4].

But untangling the role of additional tumor suppressor or oncogenic drivers of mSCLC requires the introduction of germline or conditional alleles for candidate genes into pre-existing GEMMs, a technically challenging process that can take multiple months to years. To overcome this technical chasm, the Jacks lab at MIT used a Gibson Assembly-based modular assembly platform (GMAP; a technique used to generate a series of modular vectors for bacterial and mammalian gene expression) to construct a CRISPR-mediated system for testing candidate tumor suppressor genes in mSCLC [5,6].GMAP enabled the team to construct a Cre-activated Cas9 and GFP reporter in the mSCLC model described above and an adenoviral vector, expressing a sgRNA targeting any tumor suppressor of interest and Cre recombinase.

In this way, the group developed a system where CRISPR-Cas9 and Cre-activity can be delivered to the lungs to initiate tumor formation, with active genome editing only happening in initiated tumor cells in vivo. In addition, by swapping out sgRNA sequences in the adenoviral vector using Gibson Assembly, this system can be used to quickly and simply to validate any putative tumor suppressor.
 

Many tumor models rely on in vitro models (i.e., cell lines) or in vivo animal models (i.e., GEMMs) to mimic tumor progression. However, these models have significant drawbacks to replicating the nuances of tumors in humans. PDX models, where tumors taken from a patient are engrafted into an immunocompromised mouse, can often help researchers replicate genetic, gene expression, and epigenetic characteristics of the tumor seen in the clinic [7].

One major technological barrier to using these models is that genetic perturbations of PDX models are challenging. To circumvent this difficulty, a research group from Memorial Sloan Kettering Cancer Center and Weill Cornell Medicine used Gibson Assembly to develop a lentiviral vector, called pSpCTRE, that enables tightly controlled, Dox-inducible Cas9 expression and is within the packaging size allowed by lentiviruses (a common complication when working with large Cas genes and viral vectors), resulting in high viral titers [7].

Even with robust cloning methods such as Gibson Assembly, cloning into lentiviral vectors can present problems, due to homologous recombination between long terminal repeat (LTR) sequences, necessary for integration into the host genome. When constructing lentiviral vectors, be sure to use competent E. coli cells, deficient in RecA activity (i.e., recA1 or recA13 alleles) to reduce the frequency of LTR recombination during cloning.

The researchers found, when pSpCTRE was transduced along with a sgRNA, transduced tumor cells underwent robust editing. The vector also contains a reporter gene, a truncated CD4 protein, that acts as a marker for the transduced tumor cells and as a surrogate for Cas9 expression. This inventive vector enabled efficient transduction and editing of PDX models, allowing researchers to carry out functional genomics in a clinically relevant tumor model [7].

CRISPR loci are ubiquitous across the eubacterial and archaeal kingdoms and were initially discovered for their role as an adaptive prokaryotic immune system for invading nucleic acids [8]. Part of this locus, the CRISPR array, is made up of short repeat sequences with short, unique DNA spacers. The CRISPR array is transcribed in a bacteria or archaea into a long CRISPR RNA (crRNA) and processed into individual crRNAs, each of which contains a unique spacer, capable of targeting a complementary nucleic acid sequence when complexed with a Cas protein.

CRISPR-Cas has been adapted by many labs to perform genome editing and multiplexed editing can be done by engineering in RNA processing sites [9]. However, in principle, natural CRISPR arrays could be used for genome editing and has a number of advantages: they are more compact, can be easily applied to prokaryotes that express endogenous Cas proteins, and can result in more efficient editing.

Yet, due to the presence of sequence repeats within CRISPR arrays, molecular biologists have struggled to clone and use CRISPR arrays for multiplexed editing. To address this issue, Cooper and Hasty, at UCSD, designed synthetic single-stranded oligonucleotides that bridged repeat and spacer junctions and could be efficiently annealed and ligated in the correct endogenous orientation as that found in a bacterium, Acinetobacter baylyi [10]. Following ligation, the assembled array is amplified by PCR and cloned into your vector of choice using Gibson Assembly. This strategy can be broadly applied as a robust and low-cost cloning strategy for harnessing and utilizing natural CRISPR arrays for more widespread applications.
 

As we’ve seen above, Gibson Assembly is a powerful method for constructing CRISPR vectors. So, how can you get started using it in your research?

The easiest way to get started with Gibson Assembly is to first search for, and identify, a vector that is suitable for your application. The best place to search for these, especially if you’ve spotted an attractive plasmid in a scientific publication, is on Addgene, a non-profit plasmid repository that can be accessed by other researchers. If your vector of interest hasn’t been deposited in Addgene, you can reach out to the corresponding author of a scientific publication and ask for the vectors used. There are also commercially-available CRISPR vectors that can be used in a broad range of research applications or vector selection tools that can help you figure out which are suitable for your application.

The next preparatory step is to design your primers for PCR amplification of the DNA fragments you want to stitch together using Gibson Assembly. There are many DNA editing software platforms, such as the SnapGene platform, that can be used to construct your vector in silico and design primers that bridge the fragment junctions. When designing primers, be sure that regions of complementarity have melting temperatures (T_m) of 50℃, as that is the optimal reaction temperature for Gibson Assembly. You can use a T_m calculator to check the annealing temperatures of your primers before ordering them. Following recommended protocol, PCR fragments with 20–40 nt overlapping areas are generated and mixed with Gibson Assembly reaction mix and linearized vector—for a simple one-step reaction. After incubation, reaction mix is transformed into competent E. coli cells and colonies and clones are selected and construct sequenced to confirm proper assembly.

If you need more information on designing and performing Gibson Assembly, check out our pages on GeneArt Gibson Assembly Cloning or our white paper on building large and complex Gibson Assemblies.

And if you need a simple, five-step approach to developing your own CRISPR solution, see our customized gene editing products and services.

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