Bacterial Artificial Chromosome-Based Lambda Red Recombination with the I-SceI Homing Endonuclease for Genetic Alteration of MERS-CoV

Methods Mol Biol. 2020;2099:53-68. doi: 10.1007/978-1-0716-0211-9_5.


Over the past two decades, several coronavirus (CoV) infectious clones have been engineered, allowing for the manipulation of their large viral genomes (~30 kb) using unique reverse genetic systems. These reverse genetic systems include targeted recombination, in vitro ligation, vaccinia virus vectors, and bacterial artificial chromosomes (BACs). Quickly after the identification of Middle East respiratory syndrome-CoV (MERS-CoV), both in vitro ligation and BAC-based reverse genetic technologies were engineered for MERS-CoV to study its basic biological properties, develop live-attenuated vaccines, and test antiviral drugs. Here, I will describe how lambda red recombination can be used with the MERS-CoV BAC to quickly and efficiently introduce virtually any type of genetic modification (point mutations, insertions, deletions) into the MERS-CoV genome and recover recombinant virus.

Keywords: Bacterial artificial chromosome (BAC); Coronavirus; Infectious clone; Lambda red recombination; MERS-CoV; Reverse genetics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteriophage lambda / genetics*
  • Chromosomes, Artificial, Bacterial / genetics*
  • Coronavirus Infections / prevention & control
  • Coronavirus Infections / virology*
  • Deoxyribonuclease I / genetics
  • Deoxyribonuclease I / metabolism
  • Genetic Engineering
  • Genome, Viral / genetics*
  • Homologous Recombination
  • Humans
  • Middle East Respiratory Syndrome Coronavirus / genetics*
  • Middle East Respiratory Syndrome Coronavirus / immunology
  • Mutation
  • Vaccines, Attenuated / genetics
  • Vaccinia virus / genetics
  • Viral Vaccines / genetics*


  • Vaccines, Attenuated
  • Viral Vaccines
  • Deoxyribonuclease I