Genome Editing in the Yellow Fever Mosquito Aedes aegypti using CRISPR-Cas9

Summary

Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology.

Abstract

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering field and opened the doors for precise genome editing in multiple species, including non-model organisms. In the mosquito Aedes aegypti, loss-of-function mutations and DNA insertions have been accomplished with this technology. Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology, focusing on both the generation of gene knockout and knockin lines. In this protocol, quartz needles are filled with a mixture of guide RNA, recombinant Cas9, and a plasmid containing a DNA cassette encoding a gene for a fluorescent marker, if gene knockin is desired. Embryos at the preblastoderm stage are lined up onto a strip of double-sided sticky tape placed onto a coverslip, which is subsequently mounted onto a glass slide. With the help of a microinjector, the needles are inserted gently into the posterior end of the embryos and a small volume of the CRISPR mixture is dispensed. When the embryos are hatched, the larvae are checked under the fluorescent scope, and the pupae are sex-sorted and separated in different cages. Once the adults emerge, these are reciprocally crossed with wild-type individuals, blood-fed, and placed for egg laying. Once these eggs are hatched, the fluorescent larvae collected represent individuals with stable insertion of the DNA cassette into their genome. These larvae are then grown to the adult stage, outcrossed to wild-type individuals, and then further assessed through molecular techniques to confirm that the exact sequence of the DNA cassette is present at the desired site of the mosquito genome. Homozygous lines can also be obtained by following the provided pipeline of crossing schema and molecular screening of the mutations.

Introduction

Precise genome editing has become arguably easier, but possible, with the establishment of the CRISPR-Cas technologies of molecular scissors¹. These technologies take advantage of a mechanism that the prokaryotic immune system uses to fight against phage infections². Amongst these systems, clustered, regularly interspersed, short palindromic repeats (CRISPR) along with the Cas9 nuclease usually rely upon 20 base pair RNAs, the guide RNAs (gRNAs), with sequences homologous to the targeted DNA, which are followed by an NGG protospacer adjacent motif (PAM) sequence³. The gRNAs loaded onto the Cas9 gu....

Protocol

Details related to the equipment and reagents used in this protocol are listed in the Table of Materials. All animals were handled following the Guide for the Care and Use of Laboratory Animals, as recommended by the National Institutes of Health. The procedures were approved by the UCSD Institutional Animal Care and Use Committee (IACUC, Animal Use Protocol #S17187) and UCSD Biological Use Authorization (BUA #R2401).

1. gRNAs and donor plasmid design

1. For making knockout mutants, design two gRNAs spaced by ~20-100 bp (Figure 1A).
   1. Design 20 bp gRNAs for C....

Results

Design and validation of gRNA-mediated gene targeting for HDR homology recombination
To ensure the desired gene is accurately targeted, we recommend selecting a couple of gRNAs and positioning the 5' and 3' homology arms close to the cutting site for HDR-mediated homologous recombination (Figure 1A). For example, we designed two gRNAs to target both sides of the start codon of the gene of interest and used the QF2-Hsp70-OpIE2-EC.......

Discussion

CRISPR-Cas technology has changed the landscape of genome editing by promoting target-specific changes in chromosomes¹. Even though transposable elements were essential for the generation of the first transgenic mosquitoes, their insertion sites are somewhat random, and the expression of the cargo construct (promoter + gene) may not correspond to the expression profile of the actual gene due to a genome positional effect (i.e., insertion site), which usually leads to ectopic expression

Materials

Name	Company	Catalog Number	Comments
10x Cas9 reaction buffer	PNA Bio	CB01
Benchling software	Benchling	N/A	www.benchling.com
Cas9 dilution buffer	PNA Bio	CB03
Cas9 protein	PNA Bio	CP01-50
DH5α E. coli Competent Cells	New England Biolabs	C2987
Double-sided sticky tape	Scotch Permanent	3136
Drosophila vials	Genesee Scientific	32-109
Filter papers	GE Healthcare Life Science	1450-042
Fish food	Tetra	B00025Z6YI	goldfish flakes
Flugs	Genesee Scientific	AS273
Fluorescent microscope	Leica Microsystems	M165 FC
Gene fragment	Integrated DNA Technologies	N/A
gRNA	Synthego	N/A
Halocarbon oil 700	Sigma-Aldrich	H8898
Injection microscope	Leica Microsystems	DM2000
JM109  E. coli Competent Cells	Zymo Research	T3005
Microinjector	Eppendorf		FemtoJet 4x
Microloader Tips for Filling Femtotips	Eppendorf	E5242956003
Micromanipulator	Eppendorf		TransferMan 4r
Micropipette Pullers	Sutter Instrument	P-2000
Microscope Cover Glass	Fisherbrand	12-542-B
Microscope slide	Eisco	12-550-A3
Mouse blood (live mice used for feeding)	University of California	IACUC, Animal Use Protocol #S17187	Used for mosquito blood feeding; details comply with animal ethics protocols
NEB Q5 High-Fidelity DNA polymerase	New England Biolabs	M0491S
PCR Purification Kit	Qiagen	28004
Plasmid Miniprep Kit	Zymo Research	D4036
Quartz filament	Sutter Instruments	QF100-70-10
Transcription Clean-Up Kit	Fisher Scientific	AM1908
Ultra-pure water	Life Technologies	10977-023