DNA Cloning Tips – Scarless cloning of larger DNA sequences with FastDigest Type IIS Restriction Enzymes

Discovered more than 50 years ago, restriction enzymes are foundational elements of molecular biology and genetic engineering. Restriction enzymes are DNA endonucleases (they cut DNA). They are divided into four types based on their function. For example, type I restriction enzymes like EcoKI require ATP hydrolysis for restriction and cleave at variable locations relative to the recognition site (1).

Type II restriction enzymes including EcoRI and HindIII are commonly used for cloning and other molecular biology applications since they cleave within, or at fixed positions close to the recognition site. These specific sites make it possible to cleave and ligate DNA at predictable locations. Knowledge of these sites allows the design of strategies to clone genes of interest.

There are several subtypes of type II restriction enzymes named by letter and classified by function. These functions have utility in a range of molecular biology applications. For example, methylation-dependent type IIM restriction enzymes like DpnI are often used for site directed mutagenesis. For cloning, type IIP enzymes like EcoRI and BamHI with palindromic recognition sequences, are often used. Type IIS  restrictions enzymes (BsaI, BpiI, AarI) have asymmetric recognition sites and cleave at fixed positions usually outside of the recognition sequence. This function of Type IIS enzymes is useful for the scarless cloning of larger sequences. Traditional cloning methods can add a scar or seam at the DNA joining site making that region of DNA less predictable in future experiments.
 

Cloning larger sequences using type IIP enzymes can be challenging requiring many plasmids and steps. Avoiding restriction sites within your DNA of interest becomes more difficult with longer sequences. Once a restriction site is used it is not available, so additional steps are required to introduce more sites often adding unwanted sequences to the clone. PCR cloning of larger sequences can also be challenging as errors can be introduced into the fragment. The longer the fragment the greater the chance of errors. When using PCR for the generation of DNA fragments for cloning it is important to use a high-fidelity DNA polymerase like Invitrogen Platinum SuperFi II or Thermo Scientific Phusion Plus DNA Polymerase.

The ability to shuffle is useful for many cloning applications. Parts of the protein of interest can be removed or switched with portions of DNA corresponding to functional elements of related proteins. Elements can be added or replaced that improve the functionality of the protein making it more stable, secreted, or changing post-translational modifications. Elements like glutathione S-transferase (GST) can be added to aid in purification or pull-down studies. The addition of fluorescent proteins can help track your protein or cells containing the plasmid. This is very difficult with traditional cloning methods using type IIP enzymes. To change a part of the sequence the right restriction sites need to be available at the correct location. Using these enzymes can easily result in additional nucleotides that can lead to a frame shift truncating or altering the expressed protein. Since these methods are highly specific to particular fragments and vectors, they are difficult to scale up.

To facilitate cloning of larger fragments at higher scale multiple strategies have been developed including Gibson Assembly and Golden Gate cloning.  Traditional cloning methods allow the incorporation of only up to 4 fragments compared to the Golden Gate method that permits up to 9 fragments. The Golden Gate cloning strategy allows seamless assembly of multiple fragments from several parental plasmids with high efficiency and performing DNA shuffling if fragments prepared from several homologous genes are assembled in a single reaction (2).

Golden gate assembly uses type IIS restriction enzymes which cleave outside their recognition sequences. This allows two DNA fragments with compatible restriction sites to be digested and ligated seamlessly. The resulting ligated product no longer contains the original site so it will not be redigested allowing subsequent reactions to occur. Additional fragments can be added in an orderly fashion.
 

Figure 1 illustrates this process for a single DNA fragment. Figures 2 and 3 demonstrate how different elements can be combined into the desired vector in a single restriction ligation reaction. This example also illustrates how easy it could be to replace an element like GFP with red fluorescent protein (RFP) or any other desired gene.
 

As described above Golden Gate Assembly facilitates shuffling of DNA sequences or elements in a predictable order. The use of type IIS restriction enzymes are also useful for site-directed mutagenesis. One highly efficient strategy (3) uses a PCR primer containing T7 promoter sequence and a type IIS restriction enzyme site upstream of the mutagenesis sequence generating a template for in vitro transcription. After the transcription residual DNA is digested by DNase and the RNA is used to create the desired mutant. This strategy is reported to be 100% effective in generating one or two site mutations.
 

Type II restriction enzymes are also useful for plasmid-based reverse genetics. These systems are especially useful for viral research since they can be easily manipulated to study the functional elements of the virus (4) utilized type IIS restriction enzymes to design a system to study SARS-CoV-2. Assembly of the full viral genome cDNA is challenging because of the large genomic size (~30,000 nucleotides) and toxic genomic regions. The system also requires the flexibility to study emerging mutations.

To overcome these challenges a system of seven vectors was used, each containing a type IIS restriction site. Because the sites recognize asymmetric DNA sequences and generate unique cohesive overhangs, they ensure seamless assembly into full length SARS-CoV-2. This can then be reverse transcribed to create SARS-CoV-2 RNA for further experiments. The shuffling capabilities of the system enable the introduction of tracking elements like GFP and the creation of mutant model systems.
 

Restriction enzymes continue to have significant impact on molecular biology and medicine. They are an integral part of standard cloning. However, discovery of specific properties of type IIS restriction enzymes enabled creation of more sophisticated cloning methods, such as Golden Gate cloning, and expanded usability of restriction enzymes to new applications. Type IIS restriction enzymes play an important role in the assembly of large genes. The shuffling capabilities facilitate the precise engineering of vectors. They are extremely useful for virus reengineering because they ensure directional, seamless assembly of viral fragments into genome length cDNA.

Explore new cloning capabilities that Type IIS restriction enzymes can offer, for more information please visit FastDigest Restriction Enzymes

1. Highlights of the DNA cutters: a short history of the restriction enzymes. Loenen et al. Nucleic Acids Res. 2014 Jan; 42(1):3-19.
2. cDNA Libraries: Methods and Applications, Methods in Molecular Biology, Chaofu Lu et al. (eds.), vol. 729, DOI 10.1007/978-1-61779-065-2_11, © Springer Science+Business Media, LLC 2011
3. High efficiency DNA mutagenesis mediated by using in vitro transcription, DNase I digestion, and RT-PCR. Xin W, Huang DW, Xiao H. BioTechniques. 2004 Oct;37(4):556-60.
4. Engineering SARS-CoV-2 using a reverse genetic system. Xie et al. Nature protocols. 2021 Mar;16(3):1761-84.