Abstract
CRISPR is a genome-editing platform that makes use of the bacterially-derived endonuclease Cas9 to introduce DNA double-strand breaks at precise locations in the genome using complementary guide RNAs. We developed a nuclear domain knock-in screen, whereby the insertion of a gene encoding the green fluorescent protein variant Clover is inserted by Cas9-mediated homology directed repair (HDR) within the first exon of genes that are required for the structural integrity of subnuclear domains such as the nuclear lamina and promyelocytic leukemia nuclear bodies (PML NBs). Using this approach, we compared strategies for enhancing CRISPR-mediated HDR, focusing on known genes and small molecules that impact non-homologous end joining (NHEJ) and homologous recombination (HR). Ultimately, we identified the small molecule RS-1 as a potent enhancer of CRISPR-based genome editing, enhancing HDR 3- to 6-fold depending on the locus and transfection method. We also characterized U2OS human osteosarcoma cells expressing Clover-tagged PML and demonstrate that this strategy generates cell lines with PML NBs that are structurally and functionally similar to bodies in the parental cell line. Thus, the nuclear domain knock-in screen that we describe provides a simple means of rapidly evaluating methods and small molecules that have the potential to enhance Cas9-mediated HDR.
Highlights
The recent development of systems for creating site-specific DNA double-strand breaks (DSBs) has enabled precise engineering of the mammalian genome
clustered regularly interspaced short palindromic repeats (CRISPR) reagents were designed to insert the sequence for the monomeric green fluorescent protein Clover, which is 2.5 times brighter than EGFP [31], after the second codon of the LMNA gene, which encodes the lamin A and lamin C isoforms
Homology arms corresponding to regions flanking the LMNA start codon were amplified by PCR from genomic DNA isolated from U2OS osteosarcoma cells, and were cloned into a vector flanking the Clover coding sequence (Figure 1A)
Summary
The recent development of systems for creating site-specific DNA double-strand breaks (DSBs) has enabled precise engineering of the mammalian genome. Several classes of endonucleases have been reengineered for induction of targeted DSBs in mammalian cells, including transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs) and more recently, the Streptococcus pyogenes clustered regularly interspaced short palindromic repeats (CRISPR)/Cas machinery [1,2,3]. The Cas endonuclease is directed to a locus through binding of a single guide RNA (gRNA) to its complementary genomic DNA target. To minimize potential off-target cleavage caused by association of a gRNA with multiple sites in the genome, the Cas9D10A nickase mutant can be used, which creates single-strand breaks (SSBs) instead of DSBs [7]. When the Cas9D10A nickase is expressed with two gRNAs targeting opposite strands within close proximity, a DSB will be created, while other regions targeted by each individual gRNA will only incur singlestrand nicks, which are efficiently and faithfully repaired [7]
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