Design and Validation of Guide RNAs for CRISPR–Cas9 Genome Editing in Mosquitoes

CRISPR IN MOSQUITOES AND OTHER NONTRADITIONAL MODEL ORGANISMS

CRISPR is a core component of the immune response pathway in bacteria and archaea that has been co-opted and developed into a set of precision gene editing tools that enable easier, more flexible, and more scalable means of inducing double-strand DNA (dsDNA) breaks in any organism's genome (Doudna and Charpentier 2014). Repair of these dsDNA breaks by messy processes such as nonhomologous end joining (NHEJ) can render genes nonfunctional, and more precise pathways such as homology-directed repair (HDR) can insert novel sequences into specific parts of the genome by reading from template “donor DNA” provided alongside the single-guide RNA (sgRNA) and Cas9.

In the past 10 years, CRISPR has been transformative for many fields by introducing the power of gene editing to new organisms or supercharging the speed of its application in others, such as mosquitoes, that previously relied on lower throughput protein-based technologies like zinc-finger nucleases. This tool has permitted the development of new nontraditional model species in laboratories across the world and enabled scientists to ask questions that simply cannot be modeled in the standard stable of genetic workhorses (Matthews and Vosshall 2020). In mosquitoes, CRISPR has been deployed through embryo microinjection of various combinations of CRISPR components. sgRNAs made through in vitro transcription and Cas9 provided as recombinant protein or messenger RNA (mRNA) can be injected into the developing embryo; sgRNAs can be injected into transgenic strains that express Cas9 in the developing germline (Basu et al. 2015; Dong et al. 2015; Kistler et al. 2015; Li et al. 2017; Zhu et al. 2021); or plasmids that contain regulatory sequences, which express the necessary components at the appropriate time and place to generate stable germline mutants, can be injected (Kyrou et al. 2018).

In addition to enabling mechanistic studies of basic mosquito biology, CRISPR is a key component in several genetic strategies of vector-borne disease control, such as gene drives, precision genetic sterile insect technique, and more. These strategies use CRISPR gene editing to generate transgenic mosquitoes that pass on genetic elements, making them sterile or resistant to pathogens (Wang et al. 2021).

sgRNA DESIGN AND SELECTION

The CRISPR–Cas9 system relies on RNA–DNA base-pairing interactions between the sgRNA and a complementary sequence of genomic DNA to guide the Cas9 nuclease to its specific genomic target. The recognition site for the most commonly used Streptococcus pyogenes Cas9 takes the following form: N_17–20 (NGG), with N representing any standard DNA nucleotide (A, T, C, or G). NGG is the protospacer-associated motif (PAM) and is not contained within the sgRNA DNA template or the transcribed RNA but is required to be located immediately adjacent to the target sequence in the genome for Cas9 nuclease activity.

Although one can scan the gene of interest manually when designing sgRNAs by simply looking for “GG” (or “CC”), we highly recommend the use of online sgRNA selection tools to identify potential sgRNAs. These have the benefit of scanning the rest of your organism's genome for predicted off-target activity, as well as designing primers for polymerase chain reaction (PCR) verification of mutagenesis and oligos for sgRNA synthesis. We highly recommend using CHOPCHOP (Labun et al. 2019), which has the latest versions of the common mosquito genomes loaded and ready for use, although countless other tools exist as well. In an accompanying protocol (Protocol: Validating Single-Guide RNA for Aedes aegypti Gene Editing [Lo and Matthews 2023]), we detail how to design and synthesize sgRNAs as well as how to validate sgRNA activity after injection (which is also discussed in the section sgRNA Testing here).

sgRNA TESTING

The ultimate goal of most gene editing experiments in mosquitoes is to produce stable and heritable mutant lines by editing the germline of a developing embryo. This requires that the Cas9/sgRNA complex generates a double-strand break that is repaired in a developing germ cell so that it can be passed on to the offspring of the founder. sgRNA efficiency is one variable that can influence the rate of success in creating these mutations. Once approximately three potentially suitable sgRNAs have been identified, we recommend checking their efficacy through small-scale embryo injections of sgRNA mixed with recombinant Cas9 protein, which should involve up to 50–100 embryos for each sgRNA. Large-scale injections of >500 embryos can be performed after identifying the sgRNAs that lead to the highest gene editing activities (Kistler et al. 2015). This testing is especially important if knock-ins, which have far lower rates of success in Aedes aegypti than loss-of-function NHEJ mutations, are planned (Kistler et al. 2015). Knock-ins generated by HDR are much less common than NHEJ in Ae. aegypti no matter the identity of sgRNAs used. Another option that is more commonly being used is to simply pool two to four sgRNAs into a single injection without testing, which is a strategy that has both potential benefits and caveats. On the positive side, multi-sgRNA injections appear to be very mutagenic, particularly in generating deletions between two cut sites. On the negative side, any potential off-target effects are multiplied, and structural rearrangements can occur because of multiple breaks.

ALTERNATIVES TO EMBRYO MICROINJECTION

One exciting new avenue for gene editing in nontraditional model organisms is the advent of alternatives to embryo microinjection, including receptor-mediated ovary transduction of cargo (ReMOT) control (Chaverra-Rodriguez et al. 2018; Terradas et al. 2022). This technique requires the delivery of CRISPR components directly into the ovaries of the model organism, where gene editing can happen directly in the developing embryos. In addition to providing a means of gene editing that bypasses the expensive and sometimes finicky process of embryo microinjection, adult injections can enable gene editing in animals whose eggs are difficult to collect or to inject, such as ticks (Sharma et al. 2022). However, one major limitation with ReMOT control is that there is currently no way to introduce exogenous DNA for HDR, meaning that for the time being, embryo microinjections are the best option for researchers trying to introduce exogenous DNA into their study organism's genome.