Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus.
Bothmer A, Phadke T, Barrera LA, Margulies CM, Lee CS, Buquicchio F, Moss S, Abdulkerim HS, Selleck W, Jayaram H, Myer VE, Cotta-Ramusino C.
Cells used in publication:
Tissue Origin: bone
Overall 250,000 cells were transfected using the Lonza 4D-Nucleofector (AAF-1002B 4D-Nucleofector Core unit, AAF-1002X 4D-Nucleofector X unit, SE Cell Line 4D-Nucleofector X Kit S V4XC-1032) with 200 ng of gRNA plasmid or a PCR product containing the U6 promoter/gRNA sequence/TRACR, and 750 ng of Cas9-variant plasmid (pJDS246-WT Cas9/pAF001-N863A Cas9/pJDS271-D10A Cas9 provided by K. Joung41), in the presence or absence of 50 pmol ssODN donor or 2 mg of plasmid DNA donor using the DN-100 program. For the ectopic expression of TREX2, 500 ng of plasmid AHB_26 (pcDNA3.1þTREX2-3XFLAG) was added to theCas9/gRNA/ssODN plasmid mix before nucleofection. After nucleofection, thecells were allowed to incubate at room temperature for 10 min and then resuspended in complete Dulbecco’s Modified Eagle Medium before transferring to six-well plates containing pre-warmed medium
The CRISPR-Cas9 system provides a versatile toolkit for genome engineering that can introduce various DNA lesions at specific genomic locations. However, a better understanding of the nature of these lesions and the repair pathways engaged is critical to realizing the full potential of this technology. Here we characterize the different lesions arising from each Cas9 variant and the resulting repair pathway engagement. We demonstrate that the presence and polarity of the overhang structure is a critical determinant of double-strand break repair pathway choice. Similarly, single nicks deriving from different Cas9 variants differentially activate repair: D10A but not N863A-induced nicks are repaired by homologous recombination. Finally, we demonstrate that homologous recombination is required for repairing lesions using double-stranded, but not single-stranded DNA as a template. This detailed characterization of repair pathway choice in response to CRISPR-Cas9 enables a more deterministic approach for designing research and therapeutic genome engineering strategies.
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