Pbnj Gibson Cloning for Fast Turn-Around Donor Constructs in Gene-Editing Applications

The discovery of DNA nucleases that target specific sequence signatures, such as TALE factors or CRISPR/Cas9, have significantly improved the efficiency of genome editing in vivo. To make a targeted knock-in, the target sequence in the host genome is cleaved by a nuclease and a donor template is used to repair the break. The donor templates contain designed gene expression cassettes flanked by 0.5-1 kb homologous sequences in linear or circular format. Here we report facilitating the process to make donor constructs for DNA repair using the Gibson Assembly method.

Gibson Assembly seamlessly stitches DNA fragments together without the limitation of restriction cloning. Two DNA fragments with overlapped terminal sequences will go through a step-wise enzymatic process to allow for specific recombination. These overlaps may be generated by PCR amplification using extension primers. Designing such primers and optimizing the PCR process can be labor-intensive if more than one donor construct is needed. Therefore, we exploit the use of Primer-Bridge End Joining (PBnJ) to construct donor gene expression cassettes for homology-directed repair templates. PBnJ is a variation of the Gibson Assembly reaction which bypasses the need for homologous overlaps between adjacent DNA fragments.

With a two-fold increase in turn-around time when compared to traditional Gibson Assembly, donor constructs can be effortlessly made without the need to sequence them. Our results suggest that assembly efficiency is similar to 3 fragment cloning and we can find Error-Free-Clones (EFCs) by screening 4 colonies per construct via colony PCR. In addition, it is possible to make a knock-in library with sequence verified DNA fragments and vectors. The combination of traditional Gibson Assembly and the modified Gibson Assembly PBnJ method together make a reliable tool for the construction of donor templates with minimal optimization and fast turnaround.