In this study, In-Fusion Cloning was used to quickly clone multiple, overlapping fragments of a transcriptional activation domain into a preexisting Cas9-dead viral expression vector. Insertion of this domain was done with the goal of improving the amount of transcriptional gene activation. The secondary structure of the activation domain prevented synthesis of the full sequence, and building the domain from separate pieces with traditional ligation-based methods would have limited restriction-site availability. Instead, In-Fusion technology was used to insert two synthesized portions of the domain in a single cloning reaction without any need to worry about compatible restriction sites. The full-length sequence was seamlessly cloned directly into the expression vector, and positive clones were identified by restriction digest and Sanger sequencing. The final vector was complete in three days, with hands-on time totaling just over two hours.
Speed and accuracy as well as easiness of this application have convinced me to purchase this product again."
—Christian Joerg Braun
Results
A pre-existing Cas9 expression construct was linearized by a single restriction enzyme and gel purified. Two synthesized inserts were designed with specific cloning ends that overlapped with each other and with the linearized vector. The two inserts were simultaneously cloned into the linearized expression vector, and the provided competent cells were transformed with this reaction. Colony screening was performed by restriction digest, and positive clones were further confirmed via sequencing. Out of nine colonies screened, six showed both the correct restriction digest pattern (Figure 1) and sequencing results.
I hadn't tried any other cloning methods for this experiment, but directly tested In-Fusion [Cloning]. Restriction site availability was definitely an issue with this experiment and I was happy not to depend on it."
—Christian Joerg Braun
Figure 1. Restriction digest of clones. Screening of transformant colonies was performed via EcoRI restriction digest. Results show six positive colonies out of the nine colonies tested. The three negative colonies are religated vector backbone. Data image provided by Christian Braun.
Conclusions
In Fusion technology was used to clone two synthesized pieces of a transcriptional activation domain into a Cas9 expression vector in just one cloning reaction, with a success rate of ~67%. Secondary structure of the activation domain prevented its full-length synthesis, and breaking the sequence into two parts would have introduced restriction-site availability issues that may have precluded the use of ligation-based methods. In-Fusion Cloning enabled production of a final, positive clone in three days, without the need to struggle with restriction sites or set up sequential cloning reactions.
Methods
Linearization of the preexisting Cas9 expression vector backbone was performed with EcoRI according to the manufacturer's instructions.
Two pieces of the transcriptional activation domain were synthesized (IDT) with 15-bp sequences that overlapped with each other as well as the EcoRI half-sites on the ends of the linearized expression vector (10.8 kb). These 15-bp overlaps were included to facilitate a successful In Fusion Cloning reaction. The lengths of the individual synthesized fragments were 453 bp and 1002 bp.
Figure 2. Approximation of cloning reaction setup with multiple inserts. Each synthesized fragment has 15-bp overlapping sequences required for In Fusion Cloning. The overlaps with the vector are represented by the black segments. The blue segment on Synthesized fragment 1 represents the overlap with Synthesized fragment 2. The red and blue parts of both fragments together make up the full-length transcriptional activation domain.
The smaller insert (50 ng), the larger insert (75 ng), and the linearized and purified vector backbone (50 ng) were combined in one cloning reaction with the In Fusion HD Cloning Plus enzyme mix. Reactions for a negative control (linearized vector only) and a positive control (pUC19 control vector with the control insert) were performed in parallel. Provided Stellar Competent Cells were transformed with all three reactions. Cloning and transformation were performed according to the instructions in the user manual.
The experimental plate yielded ~60 colonies, whereas the negative control plate had only three. From the experimental plate, nine colonies were screened via restriction digest with EcoRI. Agarose gel electrophoresis of the digest showed that six were positive clones; this result was then confirmed by Sanger sequencing.
