HEHR: Homing Endonuclease-Mediated Homologous Recombination for Efficient Adenovirus Genome Engineering

Abstract

Adenoviruses are non-enveloped linear double-stranded DNA viruses with over 100 types in humans. Adenovirus vectors have gained tremendous attention as gene delivery vehicles, as vaccine vectors and as oncolytic viruses. Although various methods have been used to generate adenoviral vectors, the vector-producing process remains technically challenging regarding efficacious genome modification. Based on our previously reported adenoviral genome modification streamline via linear–circular homologous recombination, we further develop an HEHR (combining Homing Endonucleases and Homologous Recombination) method to engineer adenoviral genomes more efficiently. I-PpoI, a rare endonuclease encoded by a group I intron, was introduced into the previously described ccdB counter-selection marker. We found that the I-PpoI pre-treatment of counter-selection containing parental plasmid increased the homologous recombination efficiency up to 100%. The flanking of the counter-selection marker with either single or double I-PpoI sites showed enhanced efficacy. In addition, we constructed a third counter-selection marker flanked by an alternative restriction enzyme: AbsI, which could be applied in case the I-PpoI site already existed in the transgene cassette that was previously inserted in the adenovirus genome. Together, HEHR can be applied for seamless sequence replacements, deletions and insertions. The advantages of HEHR in seamless mutagenesis will facilitate rational design of adenoviral vectors for diverse purposes.

The Adenovirus Vector Platform: Novel Insights into Rational Vector Design and Lessons Learned from the COVID-19 Vaccine

“…Based on older developments [ 32 , 33 , 34 , 35 ], advanced recombineering techniques have recently been established [ 36 , 37 , 38 ]. The desired Ad genome can not only be cloned from purified virus preparations but also from virus infected cells [ 39 ].…”

“…Furthermore, it was recently demonstrated that double-strand break generated by CRISPR/Cas9 can highly increase the homologous recombination efficiencies in mammalian genomes [ 40 ]. In concert with our observation that homologous recombination from linear fragments promoted by the full-length Rac prophage protein RecET results in substantially higher cloning efficiency, we recently developed a strategy to convert the ccdB-mediated counter selection from linear–circular homologous recombination to linear–linear homologous recombination [ 38 ].…”

ADEVO: Proof-of-concept of Adenovirus Directed EVOlution by random peptide display on the fiber knob

“…In a second step, the selection marker was removed and replaced by the desired sequence flanked by homology arms, coming either from an oligonucleotide in the case of point mutations or from PCR amplification when larger sequences needed to be inserted. This homologous recombination was performed in bacteria expressing the RecET recombinase system (26). Protocol 1 assembly backbone was obtained by replacing all HAdV-C5 sequences between the fiber gene and the right ITR with a PacI restriction site.…”