A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines

Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~ 700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very few genetic mutants have been described in mosquitoes in comparison to model organisms. The relative ease of applying CRISPR/Cas9-based gene editing has transformed genome engineering and has rapidly increased the number of available gene mutants in mosquitoes. Yet, in vivo studies may not be practical for screening large sets of mutants or possible for laboratories that lack insectaries. Thus, it would be useful to adapt CRISPR/Cas9 systems to common mosquito cell lines. In this study, we generated and characterized a mosquito optimized, plasmid-based CRISPR/Cas9 system for use in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines. We demonstrated highly efficient editing of the AGO1 locus and isolated U4.4 and Aag2 cell lines with reduced AGO1 expression. Further, we used homology-directed repair to establish knock-in Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1. These experimentally verified plasmids are versatile, cost-effective, and efficiently edit immune competent mosquito cell lines that are widely used in arbovirus studies.

Supplementary Methods (all references are numbered as in the main text)
To replace the dme U6-2 promoter with the Aae. aegypti U6 promoter (aae U6; AAEL017774 57 ), we had to alter the sgRNA cloning sites due to an internal BbsI site in the aae U6. We designed primers to add an overhang corresponding to the aae U6 to a modified sgRNA cloning site that relies on BsmBI to the pKRG2 sgRNA tracrRNA (trans-activating CRISPR RNA) scaffold and terminator sequence, with a downstream AfeI site (RU-O-22974 and RU-O-22975). The scaffold PCR and a gBlocks Gene Fragment containing the aae U6 sequence and an upstream SacI site were assembled by Gibson assembly. The assembled DNA was PCR amplified using primers RU-O-22975 and RU-O-22976. The aae U6 insert and pKRG2 were digested with SacI/AfeI, ligated, and DNA was isolated to obtain pKRG3-mU6-PUb-3xFLAG-hSpCas9, which contains the aae U6 promoter driving sgRNA expression and the aae PUb promoter driving expression of hSpCas9.
We additionally generated a version of this plasmid with the 3xFLAG at the beginning of the Cas9 removed. To remove the 3xFLAG, we introduced a NcoI site by site-directed mutagenesis using oligos RU-O-23100 and RU-O-23101. We then cloned this mutagenized insert into a clean pKRG3 background by restriction enzyme digest with BglII/XhoI. The pKRG3-NcoI plasmid was then digested with NcoI to remove the 3xFLAG and re-ligated to generate pKRG3-mU6-PUb-hSpCas9. We made another variation with the puromycin resistance cassette (pAc) added. We amplified the end of Cas9, the intervening T2A sequence 59 , and pAc from pAc-sgRNA-Cas9 30 (a gift from Ji-Long Liu; Addgene plasmid #49330; http://n2t.net/addgene:49330; RRID:Addgene_49330) using primers RU-O-23782 and RU-O-23783. We then digested pKRG3 with Eag1/BsrGI and generated pKRG3-mU6-PUb-hSpCas9-pAc by Gibson assembly. For comparative purposes, we also removed the 3xFLAG and added the pAc to hSpCas9 in pKRG3 by the same method, generating pKRG2-dU6-PUb-hSpCas9-pAc.

3xFLAG-tagged AGO1 plasmid generation
To generate overexpression plasmids as positive controls for mosquito AGO1 immunoblotting, the pKRG3 plasmid was further modified. Aag2 N-terminal 3xFLAG-tagged short and long AGO1 isoforms and the U4.4 Nterminal 3xFLAG-tagged AGO1 with pKRG3 plasmid overhangs were PCR-amplified from an in-house plasmid containing experimentally validated AGO1 sequences in each cell line (unpublished data). The hSpCas9 sequence was removed from pKRG3-mU6-PUb-hSpCas9-pAc by digestion with NcoI/BsrGI and AGO1 sequences were inserted by Gibson assembly. Alternatively, to generate an empty pKRG3 plasmid, the ends of the NcoI/BsrGI-digested plasmid were filled with T4 DNA polymerase (NEB) according to the manufacturer's protocol and blunt ends were re-ligated. Finally, the aae U6 sequence was removed from pKRG3 Ago-containing or empty plasmids by digestion with SapI/AfeIl; the ends were filled with T4 DNA polymerase and blunt ends were ligated. This generated an empty, all-purpose pKRG4-mPUb-pAc plasmid, as well as pKRG4-mPUb-3xFLAG-Aag2-AGO1-short-pAc, pKRG4-mPUb-3xFLAG-Aag2-AGO1-long-pAc, and pKRG4-mPUb-3xFLAG-U44-AGO1-pAc. To express Cre recombinase for excision of the fluorescent reporter between the loxP sites, Cre recombinase was amplified from pME66 (a gift from S. Sarbanes) using primers adding SacI/AvrII sites; the Cre insert and pKRG4-mPUb-pAc were digested with SacI/AvrII to generate pKRG4-mPUb-Cre-pAc.

CRISPR guide RNA design and cloning
We designed CRISPR RNAs (crRNAs) corresponding to AGO1. For Aag2 cells, we used the Ae. aegypti AaegL3 genome assembly, AaegL3.3 annotations, AAEL012410 (at the time of the design this was the most updated assembly; since that time the AaegL5 genome assembly has been released 23 ; the AGO1 gene ID remains the same and the updated AaegL5.2 gene annotation contains the correct AGO1 transcriptional start site). For U4.4 cells, we used the Ae. albopictus AaloF1 assembly, AaloF1.2 annotations, AALF020776. We first confirmed the genomic sequence around the experimentally determined translational start site in each cell line (unpublished data from 5'RACE and cDNA sequencing). Aag2 and U4.4 cell genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's protocol. Aag2 genomic DNA was amplified using primers RU-O-22776 and RU-O-22777, designed using the Ae. aegypti AaegL3 assembly, which has the correct annotated starting methionine. U4.4 genomic DNA was amplified using primers RU-O-22929 and RU-O-22931, designed using the Ae. albopictus AaloF1 assembly, where we could only identify a downstream methionine in 5'RACE experiments. Three guide oligos containing the BsmBI overhangs in pKRG3 plasmids were designed for each cell line based on protospacer adjacent motif (PAM) NGG sequences in close proximity to the starting methionine (RU-O-23427 to RU-O-23434, Ae. aegypti; RU-O-23456 to RU-O-23463, Ae. albopictus). The parent pKRG3-mU6-PUb-hSpCas9-pAc plasmid was digested with BsmBI and annealed oligos were ligated to generate 6 pKRG3 plasmids, one for each guide, according to protocols from Kistler et al., 2015 and Cornell's Stem Cell and Transgenic Core Facility (https://transgenics.vertebrategenomics.cornell.edu/genome-editing.html). These co-express the crRNA plus the tracrRNA as a single guide RNA (sgRNA), and hSpCas9.

Cloning of homology-directed repair (HDR) donor template
The pSL1180-HR-PUbECFP plasmid was used as the backbone for cloning an HDR donor template. A 2kb homology arm fragment around the translational start site of AGO1 in Aag2 cells was amplified from Aag2 genomic DNA using oligos RU-O-24703 and RU-O-24704, to add homology with the pSL1180-HR-PUbECFP plasmid. We ordered a gBlocks Gene Fragment containing an inserted 3xFLAG-tag between the first methionine and the second amino acid of AGO1, with silent mutations to ablate the sgRNA PAM sites. The gBlock extended past PpuMI/EagI sites in the homology arm. Next, the homology arm was digested with PpuMI/EagI to drop out the central ~160 nt, generating 2 ~1kb homology fragments overlapping the gBlock. pSL1180-HR-PUbECFP was digested with NotI/EcoRI, dropping out the PUB-eCFP, and the fragments were assembled to generate the intermediate plasmid pSL1180-HR-Aag2-3xFLAG-AGO1. Next, pSL1180-HR-Aag2-3xFLAG-AGO1 was modified to add the loxp-PUb-RFP-loxP cassette, with overlaps corresponding to the upstream homology arm and downstream 3xFLAG-AGO1 sequence. We generated four PCRs:   (a) Immunoblot of AGO1 showed Aag2 clones with wild-type (WT) and reduced (salmon arrows) AGO1 protein levels; A-E denote clones obtained from each sgRNA singly or in combination; ns = not shown (in reporter assay).