Knock-In of a 25-Kilobase Pair BAC-Derived Donor Molecule by Traditional and CRISPR/Cas9-Stimulated Homologous Recombination

Abstract

ABSTRACTHere, we describe an expansion of the DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus. Driving our efforts was the desire to create a whole animal model that would replace 17 kbp of the mouseBcl2l11gene with the corresponding 25-kbp segment of humanBCL2L11, including a conditionally removable segment (2.9-kbp) of intron 2, a cryptic human exon immediately 3′ of this, and a native human exon some 20 kbp downstream. Using two methods, we first carried out the replacement by employing a combination of bacterial artificial chromosome recombineering, classic ES cell targeting, dual selection, and recombinase-driven cassette removal (traditional approach). Using a unique second method, we employed the same vector (devoid of its selectable marker cassettes), microinjecting it along with CRISPR RNA guides andCas9into mouse zygotes (CRISPR approach). In both instances we were able to achieve humanization ofBcl2l11to the extent designed, remove all selection cassettes, and demonstrate the functionality of the conditionally removable,loxP-flanked, 2.9-kbp intronic segment.AUTHOR SUMMARYClustered regularly interspaced short palindromic repeat (CRISPR) technology can be used to place DNA sequences (designed in the laboratory) into the genomes of living organisms. Here, we describe a new method, whereby we have replaced an exceptionally large segment of the mouseBcl2l11gene with the corresponding segment of humanBCL2L11gene. The method represents an expansion of the DNA size limitations typically associated with the introduction of DNA sequences through traditional CRISPR methods.