Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease.
Sequence-specific endonucleases recognizing long target sequences are emerging as powerful tools for genome engineering. These endonucleases could be used to correct deleterious mutations or to inactivate viruses, in a new approach to molecular medicine. However, such applications are highly demanding in terms of safety. Mutations in the human RAG1 gene cause severe combined immunodeficiency (SCID). Using the I-CreI dimeric LAGLIDADG meganuclease as a scaffold, we describe here the engineering of a series of endonucleases cleaving the human RAG1 gene, including obligate heterodimers and single-chain molecules. We show that a novel single-chain design, in which two different monomers are linked to form a single molecule, can induce high levels of recombination while safeguarding more effectively against potential genotoxicity. We provide here the first demonstration that an engineered meganuclease can induce targeted recombination at an endogenous locus in up to 6% of transfected human cells. These properties rank this new generation of endonucleases among the best molecular scissors available for genome surgery strategies, potentially avoiding the deleterious effects of previous gene therapy approaches.
Grizot
Sylvestre
S
Cellectis SA, Cellectis Genome Surgery, 93235 Romainville, France.
Smith
Julianne
J
Daboussi
Fayza
F
Prieto
Jesús
J
Redondo
Pilar
P
Merino
Nekane
N
Villate
Maider
M
Thomas
Séverine
S
Lemaire
Laetitia
L
Montoya
Guillermo
G
Blanco
Francisco J
FJ
Pâques
Frédéric
F
Duchateau
Philippe
P
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019281:Dimerization; D018390:Gene Targeting; D016654:Genes, RAG-1; D005818:Genetic Engineering; D006801:Humans; D011995:Recombination, Genetic; D016511:Severe Combined Immunodeficiency / Q000235:genetics
D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI