Strategies to improve homology-based repair outcomes following CRISPR-based gene editing in mosquitoes: lessons in how to keep any repair disruptions local

Abstract

Programmable gene editing systems such as CRISPR-Cas have made mosquito genome engineering more practical and accessible, catalyzing the development of cutting-edge genetic methods of disease vector control. This progress, however, has been limited by the low efficiency of homology-directed repair (HDR)-based sequence integration at DNA double-strand breaks (DSBs) and a lack of understanding about DSB repair in mosquitoes. Innovative efforts to optimize HDR sequence integration by inhibiting non-homologous end joining or promoting HDR have been performed in mammalian systems, however many of these approaches have not been applied to mosquitoes. Here, we review some of the most relevant steps of DNA DSB repair choice and highlight promising approaches that influence this choice to enhance HDR in the context of mosquito gene editing.

Keywords: Aedes; CRISPR; DNA repair; Gene editing; Homology-dependent repair; Mosquito.

Fig. 1.

53BP1 influences repair pathway choice. A DSB which has had initial repair factors recruited (MRN complex, ATM, MDC1) and adjacent to a nucleosome with ubiquitylated Lys15 of histone 2A (H2AK15ub) and mono- or di-methylated Lys20 of histone 4 (H4K20me1/2). B In G1 phase, 53BP1 binds to H2A15ub and H4K20me1/2 via its UDR motif and tudor domain respectively. RIF1 is recruited to 53BP1 via binding to ATM-phosphorylated residues. BRCA1 foci formation is inhibited in G1 via 53BP1 and RIF1, where the N terminus ATM target sites in 53BP1 are necessary for its ability to recruit and interact with RIF1 and PTIP. ATM-phosphorylation of 53BP1 leads to recruitment of other NHEJ-promoting factors such as PTIP and EXPAND1, and leads to blocking of end resection and promotion of NHEJ. C In G2/S phases, CtIP is recruited by and bound to the MRN complex. After CtIP is phosphorylated, BRCA1 binds, 53BP1-RIF1 are inhibited from binding to chromatin, and end resection and HDR are promoted. For all panels, colored fill indicates protein factors are conserved in vector mosquiotes, while while fill (CtIP, BRCA1) indicates repairs factors that appear to be absent in mosquitoes

Fig. 2

Modified nuclease systems to bias HDR at specific DSBs. A Schematic of HDR factors tethered to Cas9 via peptide linker can promote strand-invasion (Cas9-yRAD2, Cas9-Brex27), end-resection (Cas9-CtIP, Cas9-MRE11, Cas9-UL12), or single-strand annealing (Cas9-RecA) at a DNA double-strand break. B Diagram of the REDIT system composed of Cas9, sgRNA, RNA aptamer with MS2 loop, MS2 coat protein (MCP), RecT, and either single-strand DNA (ssDNA) or double-stranded DNA (dsDNA) donors. C Visual representation of Cas9-hGem levels during cell cycle stages and Cas9-hGem construct. D Overview of S1mplex components: Cas9, sgRNA, RNA aptamer, streptavidin, biotin, and either ssDNA or dsDNA donors. E Cas9 tethered to PCV, an HUH endonuclease forming a covalent bond with ssODN donor. F A transcription factor DNA binding domain fused to Cas9 with a peptide linker binding to motifs presents donor DNA