Modulating DNA Repair Pathways to Improve Precision Genome Engineering

Abstract

Programmable nucleases like the popular CRISPR/Cas9 system allow for precision genome engineering by inducing a site-specific DNA double strand break (DSB) within a genome. The DSB is repaired by endogenous DNA repair pathways, either nonhomologous end joining (NHEJ) or homology directed repair (HDR). The predominant and error-prone NHEJ pathway often results in small nucleotide insertions or deletions that can be used to construct knockout alleles. Alternatively, HDR activity can result in precise modification incorporating exogenous DNA fragments into the cut site. However, genetic recombination in mammalian systems through the HDR pathway is an inefficient process and requires cumbersome laboratory methods to identify the desired accurate insertion events. This is further compromised by the activity of the competing DNA repair pathway, NHEJ, which repairs the majority of nuclease induced DNA DSBs and also is responsible for mutagenic insertion and deletion events at off-target locations throughout the genome. Various methodologies have been developed to increase the efficiency of designer nuclease-based HDR mediated gene editing. Here, we review these advances toward modulating the activities of the two critical DNA repair pathways, HDR and NHEJ, to enhance precision genome engineering.

Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery

“…120 With ZFNs, TALENs, and CRISPR-Cas, editing occurs after cellular DNA repair pathways resolve the DNA double-stranded break (DSB) by non-homologous end joining (NHEJ), which can introduce insertions or deletions, or by homology-directed repair (HDR), with a donor sequence present at the site of the break. 121 ZFNs and TALENs facilitate sequence recognition by protein-DNA interactions; however, complicated protein engineering required to create specific DNA-targeting protein domains restricts their broad application. 112,113 The discovery of RNA-guided DNA endonucleases, such as CRISPR-Cas9, Cpf1 (Cas12a), and Cas12b, equipped scientists with a relatively easy-to-use platform to alter genomic information.…”

A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

“…The identified sites could also be used to display inhibitors of DNA repair pathways for their local inhibition at the site of the Cas9-induced double-strand break. For example, co-administration of Cas9 with small-molecule inhibitors of the NHEJ pathway can enhance precision editing, 11 but concerns about mutagenesis stemming from genomewide NHEJ inhibition has limited the utility of such inhibitors. 38,39 Local NHEJ-pathway inhibition at the strand break site through the multivalent display of NHEJ inhibitors on Cas9 itself may allay such concerns.…”

“…10 Multivalent display of DNA repair pathway inhibitors (e.g., NHEJ or p53 pathway inhibitors) or cell-specific ligands could also enhance precision and efficacious genome editing. 11,12 An ideal conjugation platform for Cas9 should have several characteristics. First, the platform should be compatible with a diverse set of cargoes (e.g., small molecules, nucleic acids, nanoparticles, antibodies, PEG chains) and allow their multivalent display.…”

DNA barcoding reveals that injected transgenes are predominantly processed by homologous recombination in mouse zygote

“…With the development of CRISPR-based genome editing, the focus has shifted to other roles of DNA repair pathways: HR is exploited for precise genetic modifications, such as transgene knock-ins, and NHEJ is used to knock-out genes (7,8). The obvious importance of these pathways for the field of genome editing serves as a driver for studying activity of these pathways in different cells (9,10); understanding crosstalk between NHEJ and HR (11); and for developing new methods for the preferential activation of a particular pathway (12). Modification of the genome at the zygote stage is an advantageous method for obtaining genetically modified animals.…”