Understanding the DNA damage response in order to achieve desired gene editing outcomes in mosquitoes

Abstract

Mosquitoes are high-impact disease vectors with the capacity to transmit pathogenic agents that cause diseases such as malaria, yellow fever, chikungunya, and dengue. Continued growth in knowledge of genetic, molecular, and physiological pathways in mosquitoes allows for the development of novel control methods and for the continued optimization of existing ones. The emergence of site-specific nucleases as genomic engineering tools promises to expedite research of crucial biological pathways in these disease vectors. The utilization of these nucleases in a more precise and efficient manner is dependent upon knowledge and manipulation of the DNA repair pathways utilized by the mosquito. While progress has been made in deciphering DNA repair pathways in some model systems, research into the nature of the hierarchy of mosquito DNA repair pathways, as well as in mechanistic differences that may exist, is needed. In this review, we will describe progress in the use of site-specific nucleases in mosquitoes, along with the hierarchy of DNA repair in the context of mosquito chromosomal organization and structure, and how this knowledge may be manipulated to achieve precise chromosomal engineering in mosquitoes.

CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity

“…The type II CRISPR/Cas9 endonuclease system evolved in bacteria as a defensive measure against viruses to cleave invading foreign DNA ( Barrangou et al, 2007 ). This natural defense system has been engineered into a bipartite genome editing tool, consisting of a guide RNA (gRNA) that directs double stranded DNA cleavage by the Cas9 endonuclease at specific locations within the genome ( Jinek et al, 2012 ) and has been adapted for use in a myriad of organisms (reviewed in: ( Bassett and Liu, 2014 ; Doudna and Charpentier, 2014 ; Hsu et al, 2014 ; Overcash et al, 2015 ; Sander and Joung, 2014 ; Sternberg and Doudna, 2015 ; Zhang et al, 2014 ), including Drosophila ( Bassett et al, 2013 ; Gratz et al, 2013a ; Kondo and Ueda, 2013 ; Ren et al, 2013 ; Yu et al, 2013 ). Following gRNA/Cas9 cleavage at a given target site, mutations can be generated at that genomic location via one of two pathways: error-prone non-homologous end joining (NHEJ) DNA repair, which typically generates small insertions and/or deletions (indels) at the cut site, or the more precise homology-directed repair (HDR) pathway that copies sequences from a homologous DNA template ( Gratz et al, 2013 ).…”

Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9

“…aegypti [ 30 ]. Other repair mechanisms such as alternative non-homologous end joining (A-NHEJ) or single-strand annealing (SSA) can also play a role [ 58 ]. The NHEJ pathway is an error-prone mechanism that cause gene disruption by introducing insertions or deletions, whereas for HDR, genes are replaced by recombination and a homologous sequence is required.…”

The dawn of active genetics

“…[1][2][3][4]) has proven to be a highly effective genome editing tool in a wide variety of organisms including diverse animals, plants, and yeast (reviewed in Refs. [5][6][7][8][9][10][11]) ( Fig. 1A and B).…”