In vivoblunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining

Abstract

The CRISPR/Cas9 system facilitates precise DNA modifications by generating RNA-guided blunt-ended double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting these breaks to insert exogenous PCR-generated sequences in a homology-independent manner without loss of additional nucleotides. This method is useful for making precise additions to the genome such as insertions of marker gene cassettes or functional elements, without the need for homology arms. We successfully utilized this method in human and mouse cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 36% in HEK293 cells without selection. We also created versions of Cas9 fused to the FKBP12-L106P destabilization domain in an effort to improve Cas9 performance. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant increase the repertoire of precision genome engineering approaches.

Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells

“…(2015) pioneered an experiment for replacing human THY1 gene with its mouse homolog through double gRNAs/Cas9-mediated cleavage in hiPSCs, they also found DNA-fragment deletion in between the two cleaved sites and rejoining of the blunt ends without indels. This precise NHEJ or precise gene deletion after paired gRNA-mediated DSBs has also been observed in HEK293 cells and hematopoietic stem cells ( Geisinger et al., 2016 , Ho et al., 2015 , Mandal et al., 2014 , Zheng et al., 2014 ). Given these predictable virtues of DNA cleavage and repair paradigms mediated by double gRNAs, the current paired-KO strategy is suitable for gene knockout in hPSCs without generating any non-natural proteins.…”

“…The 15- to 23-bp-long complementary sequence binds to the target genomic locus through strict Watson-Crick pairing ( Fu et al., 2014 , Hsu et al., 2013 , Stemmer et al., 2015 ), where the gRNA directs Cas9 for DNA cleavage and therefore results in a DSB at a desired site ( Jinek et al., 2012 ). A DSB introduced into the genome will initiate DNA repair through either error-prone non-homologous end joining (NHEJ) or homology-directed repair (HDR) in the presence of exogenous DNA templates ( Geisinger et al., 2016 , Heyer et al., 2010 , Jasin and Rothstein, 2013 , Lackner et al., 2015 ).…”

Decoding non-random mutational signatures at Cas9 targeted sites

“…Our results suggest that incorporation of staggered rather than blunt oligo-duplexes into the broken ends may occur more efficiently and therefore improve techniques such as GUIDE-seq to allow a more efficient, sensitive and precise detection of Cas9 off-targets. Similarly, these findings could be useful for designing more efficient NHEJ-based knock-in strategies by exploiting high efficiency ligation between the staggered ends of a Cas9 targeted transgene and target endogenous loci ( 74 , 75 ).…”

Repair of a Site-Specific DNA Cleavage: Old-School Lessons for Cas9-Mediated Gene Editing

“…This can be accomplished through the use of an auxin-inducible degron 108 , as has also been done for the site-specific AsiSI endonuclease in mammalian cells 109, 110 . Another way to destabilize Cas9 has been to fuse it to the FKBP12-L106P domain 100 . In this way, one can observe in budding yeast the cleaved ends re-join in real time, as was first done with HO.…”