Scarless Cloning with Golden Gate Assembly

Type II restriction enzymes, including Thermo Scientific FastDigest EcoRI and Thermo Scientific FastDigest HindIII, are commonly used for cloning and other molecular biology applications since they cleave within, or at fixed positions close to the recognition sites. 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 are used in a wide 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 restriction enzymes (Thermo Scientific Eco31I (BsaI), Thermo Scientific FastDigest Bpil (BbsI), Thermo Scientific 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. When cloning multiple fragments, traditional cloning methods can add a scar or seam, which are additional nucleotides at the DNA fragments joining site. Regions with scars can make the DNA less predictable in downstream experiments. Scarless cloning allows creating precise DNA constructs without additional nucleotides.

Scarless cloning allows shuffling of multiple DNA fragments. 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 to enhance the functionality of the protein, such as increasing its stability, improving its secretion, or altering its 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 proteins or cells containing the plasmid.

Cloning larger sequences using type IIP enzymes can be challenging and face several challenges:

• Limited availability of unique restriction sites: Avoiding restriction sites within longer DNA sequences of interest is relatively difficult. This limitation poses challenges to isolating and cloning the desired fragment without cutting the sequence in unintended ways. Additionally, once a restriction site is used, it is unavailable; so, additional steps are required to introduce more sites often adding unwanted sequences in the clone.
• DNA fragment stability: Large DNA fragments are more prone to degradation and instability during the cloning process. Handling and maintaining the integrity of large fragments can be challenging, resulting in reduced cloning success and higher rates of DNA breakage.
• Unintended sequences or disruption to coding sequence: The recognition sites of Type IIP restriction enzymes are retained in the final construct, resulting in extra base pairs at the junctions of the cloned fragments. These additions can be a few base pairs long and may disrupt open reading frames (ORFs) if they occur within coding sequences, potentially leading to frameshift mutations or the introduction of unintended amino acids. 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.
• Increased complexity of ligation: Ligation efficiency decreases with the size of the DNA fragment as larger fragments are more prone to incomplete ligation, leading to lower cloning efficiency and higher rates of vector recircularization without the insert.
• Limited scalability: Traditional restriction enzyme-based cloning methods are often optimized for small-scale laboratory experiments. Scaling up these methods to handle large volumes or high-throughput applications can be difficult and inefficient.

To facilitate cloning of larger fragments at higher scale, multiple strategies have been developed, including Gibson Assembly cloning, Golden Gate cloning, and CRISPR-Cas9 mediated cloning.

Golden Gate cloning is a scarless cloning method that allows multiple DNA fragments to be assembled into a vector in a single reaction. The Type IIS restriction enzymes, which cut the DNA downstream from their recognition sites, can be used to generate DNA fragments with distinct overhangs. Assembly of digested fragments then proceeds through the annealing of complementary four-base overhangs on adjacent fragments. The digested fragments and the final assembly no longer contain Type IIS restriction enzyme recognition sites, because these recognition sites where the enzyme had bound were upstream of the cleavage, so it will not be redigested, allowing digestion and ligation to be carried out simultaneously.

Hierarchical assembly in Golden Gate cloning is a strategy used to construct large and complex DNA sequences by assembling smaller DNA fragments in a stepwise manner. This approach allows for the precise and efficient construction of multi-part genetic constructs without leaving any unwanted sequences (scars).

When preparing for hierarchical assembly, target DNA sequence is divided into smaller, manageable fragments. Each fragment is individually cloned into vectors using techniques such as Gibson Assembly or Golden Gate Assembly.

Once the smaller fragments are successfully cloned and verified, they are sequentially combined in subsequent rounds of assembly to build the final, larger DNA sequence.

Golden Gate cloning is an efficient and versatile method for DNA assembly that offers several significant advantages over traditional cloning techniques:

• Seamless assembly: Golden Gate method utilizes type IIS restriction enzymes that cut DNA outside of their recognition sites, thus allowing precise and seamless assembly of DNA fragments without leaving any unwanted sequences (scars) at the junctions.
• Multi-fragment assembly: While traditional cloning methods typically allow the incorporation of only up to 4 fragments, Golden Gate cloning has been used to assemble varying numbers of DNA parts, with reports of successfully assembling as many as 52 fragments [5].
• High efficiency: The method’s ability to perform simultaneous digestion and ligation in a single reaction tube enhances the efficiency of the cloning process. This reduces the number of steps and minimizes the potential for errors or contamination.
• Flexibility in design: Researchers can design custom overhangs for the DNA fragments, allowing for specific and directed assembly. This flexibility enables the creation of diverse genetic constructs with precise control over the order and orientation of the fragments.
• Wide adoption and support: Based on Golden Gate assembly, various systems like MoClo, Golden Braid, and Loop assembly have been developed following a common syntax concept—standardized set of rules and sequences used to ensure that DNA parts can be assembled in a predictable and efficient manner. This standardization ensures that various genetic parts—such as promoters, coding sequences, and terminators—can be seamlessly combined in a predictable manner. This facilitates the creation of complex genetic constructs, enhances compatibility and reusability of parts, and streamlines the cloning process, making Golden Gate cloning a powerful and versatile tool in molecular biology and synthetic biology [2].

Golden Gate cloning is a powerful tool in molecular biology due to its efficiency, precision, and versatility, enabling researchers to construct complex DNA sequences with ease. Golden Gate cloning has expanded the usability of restriction enzymes to new applications such as:

• Synthetic biology and metabolic pathway engineering to construct synthetic metabolic pathways by assembling multiple genes and regulatory elements into a single plasmid [6]
• Viral vector construction to create viral vectors for gene therapy and vaccine development by assembling multiple genetic elements into a single construct [8]
• Plant genetic engineering to create complex genetic constructs for plant transformation, enabling the study and manipulation of plant genes and pathways [7]
• CRISPR-Cas9 genome editing to construct CRISPR-Cas9 vectors by assembling guide RNA sequences and Cas9 coding regions, facilitating precise genome editing in various organisms [9]

For new cloning capabilities by Type IIS restriction enzymes, explore FastDigest restriction enzymes

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 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 [3]. After 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.

Golden Gate cloning is 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 utilized type IIS restriction enzymes to design a system to study SARS-CoV-2 [4]. 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 genome. 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.

Golden Gate cloning is favored in plant genetic engineering due to its high efficiency, precision, modularity, and versatility. The technique's ability to seamlessly assemble multiple DNA fragments in a single reaction, coupled with its compatibility with automation and high-throughput applications, makes it an ideal choice for constructing complex genetic constructs.

Golden Gate cloning leverages the capabilities of Type IIS enzymes to streamline the process of genetic modification. By adhering to a common syntax, researchers can ensure that various genetic elements—such as promoters, coding sequences, terminators, and regulatory sequences—are compatible and can be easily combined. 

Type IIS restriction enzymes in plant engineering:

Eco31I (BsaI) - Recognition Sequence: GGTCTC(1/5)^ 

Esp3I (BsmBI) - Recognition Sequence: CGTCTC(1/5)^ 

BpiI (BbsI) - Recognition Sequence: GAAGAC(2/6)^

AarI – Recognition Sequence: CACCTGC(4/8)^

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2. Chaofu L, John B, James WG, editors (2011) cDNA Libraries: Methods and Applications, Methods in Molecular Biology. Springer Protocols: Humana Press.
3. Xin W, Huang DW, Xiao H (2004) High efficiency DNA mutagenesis mediated by using in vitro transcription, DNase I digestion, and RT-PCR. BioTechniques 37(4):556–560. doi: 10.2144/04374BM06.
4. Xie X, Lokugamage KG, Zhang X, et al. (2021) Engineering SARS-CoV-2 using a reverse genetic system. Nature protocols 16(3):1761–1784.
5. Pryor JM, Potapov V, Bilotti K, et al. (2022) Rapid 40 kb genome construction from 52 parts through data-optimized assembly design. ACS Synth Biol 11(6):2036–2042. doi: 10.1021/acssynbio.1c00525.
6. Engler C, Gruetzner R, Kandzia R, et al. (2009) Golden Gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE 4(5). doi: 10.1371/journal.pone.0005553.
7. Weber E, Engler C, Gruetzner R, et al. (2011) A modular cloning system for standardized assembly of multigene constructs. PLoS ONE 6(2). doi: 10.1371/journal.pone.0016765.
8. Bilotti K, Keep S, Sikkema AP, et al. (2024) One-pot Golden Gate assembly of an avian infectious bronchitis virus reverse genetics system. PLoS ONE 19(7). doi: 10.1371/journal.pone.0307655.
9. Vad-Nielsen J, Lin L, Bolund L, et al. (2016) Golden Gate assembly of CRISPR gRNA expression array for simultaneously targeting multiple genes. Cell Mol Life Sci 73(3):4315–4325. doi: 10.1007/s00018-016-2271-5.