Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti Sanjay Basua,1, Azadeh Aryana,1, Justin M. Overcasha, Glady Hazitha Samuela, Michelle A. E. Andersona, Timothy J. Dahlemb, Kevin M. Mylesa,2, and Zach N. Adelmana,2 aFralin Life Science Institute and Department of Entomology, Virginia Tech, Blacksburg, VA 24061; and bMutation Generation and Detection Core, Health Science Center Core Research Facility, University of Utah, Salt Lake City, UT 84132 Edited by Anthony A. James, University of California, Irvine, CA, and approved February 24, 2015 (received for review February 6, 2015) Conventional control strategies for mosquito-borne pathogens range of potential target sequences, already leading to the pro- such as malaria and dengue are now being complemented by the posal of alternative gene drive designs and regulatory structure development of transgenic mosquito strains reprogrammed to based on these more facile genetic tools (5, 6). Identified in type generate beneficial phenotypes such as conditional sterility II Streptococcus pyogenes as a heritable immune defense mech- or pathogen resistance. The widespread success of site-specific anism against viruses and plasmids (7), endogenous CRISPR loci nucleases such as transcription activator-like effector nucleases are comprised of 30–40 nucleotide (nt) variable sequences sep- (TALENs) and clustered regularly interspaced short palindromic arated by short direct repeats. CRISPR locus transcripts are used repeats (CRISPR)/Cas9 in model organisms also suggests that re- to generate short CRISPR RNAs (crRNAs) that act in con- programmable gene drive systems based on these nucleases may junction with a common transactivating-RNA (trRNA) to guide be capable of spreading such beneficial phenotypes in wild mos- Cas9 endonuclease to the complementary double-stranded DNA quito populations. Using the mosquito Aedes aegypti, we deter- (dsDNA) target site. An advancement in this technique has led mined that mutations in the FokI domain used in TALENs to to coupling of the crRNA and trRNA into a single chimeric generate obligate heterodimeric complexes substantially and sig- guide RNA (8), resulting in a two-component system to cleave an nificantly reduce gene editing rates. We found that CRISPR/Cas9- 18- to 20-nt target sequence. However, the effectiveness of the based editing in the mosquito Ae. aegypti is also highly variable, CRISPR/Cas9 system in disease vector mosquitoes has not yet with the majority of guide RNAs unable to generate detectable been evaluated. editing. By first evaluating candidate guide RNAs using a transient Transcription activator-like effector nucleases (TALENs) are embryo assay, we were able to rapidly identify highly effective also a two-component site-specific nuclease system and have guide RNAs; focusing germ line-based experiments only on this been used in genomic editing of a number of different genomes cohort resulted in consistently high editing rates of 24–90%. Mi- including the mosquitoes Ae. aegypti (9) and Anopheles gambiae croinjection of double-stranded RNAs targeting ku70 or lig4, both (10). TALENs are hybrid proteins consisting of a TAL DNA essential components of the end-joining response, increased binding domain (discovered in the plant pathogenic bacteria recombination-based repair in early embryos as determined by plas- Xanthomonas) and the FokI nuclease domain (11). An individual mid-based reporters. RNAi-based suppression of Ku70 concurrent TALEN consists of a left and right monomer protein, each with with embryonic microinjection of site-specific nucleases yielded its own FokI nuclease domain that, on dimerization, induces consistent gene insertion frequencies of 2–3%, similar to tradi- tional transposon- or ΦC31-based integration methods but with- Significance out the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and Mosquitoes are vectors of both parasites and viruses re- simplify the evaluation of novel Cas9-based gene drive systems. sponsible for high-impact diseases including malaria, den- gue, and chikungunya. Novel interventions based on genetic Aedes | CRISPR | TALEN | transgenic | recombination modification of the mosquito genome are currently being de- veloped and implemented. To comprehensively exploit such interventions, detailed knowledge of mosquito physiology, osquitoes transmit disease agents that cause malaria, dengue genetics, and genome engineering are required. We developed Mfever, chikungunya, and more. Conventional control strate- and validated a two-step process for performing high-efficiency gies based on insecticides, bed nets, source reduction, vaccines, site-specific insertion of genetic material into the mosquito ge- and drug treatments are now being complemented by transgenic nome by first evaluating candidate site-specific nucleases in a approaches, which may soon include pathogen-resistant mos- rapid format, followed by germ line-based editing where the quito strains. Although development of these strategies is on- choice of DNA repair response is constrained. This model should going, field releases of first-generation technologies have had significantly accelerate both basic and applied research con- some remarkable successes (1, 2). Significant effort is currently cerning disease vector mosquitoes. being expended to develop transgene-based approaches with so- “ ” called gene drive systems designed to increase the incidence of Author contributions: S.B., A.A., J.M.O., G.H.S., T.J.D., K.M.M., and Z.N.A. designed re- inheriting specific alleles in a target population (3). Although search; S.B., A.A., J.M.O., G.H.S., and M.A.E.A. performed research; T.J.D. contributed new a number of theoretical systems have been proposed, only one reagents/analytic tools; S.B., A.A., J.M.O., G.H.S., M.A.E.A., T.J.D., K.M.M., and Z.N.A. based on the site-specific homing endonuclease I-SceI has been analyzed data; and S.B., A.A., T.J.D., K.M.M., and Z.N.A. wrote the paper. shown to successfully drive a genetic modification into a mos- The authors declare no conflict of interest. quito population in large cage studies (4). This article is a PNAS Direct Submission. Unlike homing endonucleases, which are difficult to re-engineer, 1S.B. and A.A. contributed equally to this work. site-specific nucleases based on the type II clustered regularly 2To whom correspondence may be addressed. Email: [email protected] or [email protected]. interspaced short palindromic repeats (CRISPR)/Cas9 are un- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. rivaled in the ease with which they can be tailored to a wide 1073/pnas.1502370112/-/DCSupplemental. 4038–4043 | PNAS | March 31, 2015 | vol. 112 | no. 13 www.pnas.org/cgi/doi/10.1073/pnas.1502370112 Downloaded by guest on September 25, 2021 Fig. 1. Heterodimeric TALEN activity in mosquito embryos. (A) Representation of the TALEN con- structs used to target Ae. aegypti kmo. All pos- sessed the same set of TALE repeats with the exception of the final 1/2 repeat, which either perfect-matched (red) or was mismatched (blue) to the target site (block arrow). Constructs also dif- fered in the FokI domain as indicated. Architecture used in ref. 9 is indicated (#). (B) SSA assay to detect TALEN activity in mosquito embryos. Each point represents a group of ∼100 injected embryos; mean and SD are indicated. Groups were found to be significantly different by ANOVA (P < 0.05), with assignment to groups (a, b) by Dunnett’s multiple comparison test. (C) HRMA-based analysis of amplicons obtained from mosquito embryo DNA 24 h following injection with the indicated kmo TALEN pair [MM (mismatched) and PM (perfect match)] or from noninjected controls (CNT). dsDNA breaks. Initially, WT versions of the FokI domain were of initial transgenic technology, this two-step HDR approach used in TALEN architectures; however, activity at off-target sites should dramatically accelerate efforts to engineer mosquito was attributed to the homodimerization of left or right TALEN genomes for genetic control strategies, as well as enable inves- monomers (left-left or right-right) (12). Targeted mutations of the tigations into the basic biology of important vector species. FokI dimerization plane, R487D (FokI-DD) or D483R (FokI-RR), established a requirement for heterodimerization of the TALEN Results left and right protein monomers that significantly reduced off-target Although TALEN-mediated gene editing has been demon- activity (13, 14). However, these improvements also decreased tar- strated in mosquitoes (9, 10), the influence of different TALEN get activity by reducing dimerization energy (15). Introduction of architectures on the induction of DSBs has not been examined. secondary mutations that added oppositely charged amino acids to We generated six TALEN pairs, targeting genes related to each version, R487D and N496D (FokI-DDD) or D483R and mosquito immunity (dcr2, ago2) or DSB repair (lig4) pathways. H537R (FokI-RRR), restored activity to near WT levels while However, following injection of embryos, we failed to recover still maintaining decreased off-target activity (15). The efficacy any heritable gene editing events (dcr2, ago2; Table S1) or evi- of each of the different TALEN architectures (WT vs. DD/RR dence of nuclease activity in transient embryos assays (dcr2, ago2, vs. DDD/RRR) has