In vivo selection of engineered homing endonucleases using double-strand break induced homologous recombination.
Homing endonucleases, endonucleases capable of recognizing long DNA sequences, have been shown to be a tool of choice for precise and efficient genome engineering. Consequently, the possibility to engineer novel endonucleases with tailored specificities is under strong investigation. In this report, we present a simple and efficient method to select meganucleases from libraries of variants, based on their cleavage properties. The method has the advantage of directly selecting for the ability to induce double-strand break induced homologous recombination in a eukaryotic environment. Model selections demonstrated high levels of enrichments. Moreover, this method compared favorably with phage display for enrichment of active mutants from a mutant library. This approach makes possible the exploration of large sequence spaces and thereby represents a valuable tool for genome engineering.
Chames
Patrick
P
CELLECTIS S.A., 102 route de Noisy 93235, Romainville, France.
Epinat
Jean-Charles
JC
Guillier
Sophie
S
Patin
Amélie
A
Lacroix
Emmanuel
E
Pâques
Frédéric
F
D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D023281:Genomics; D009154:Mutation; D019151:Peptide Library; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics
D019151:Peptide Library; D004247:DNA; D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI