Research Article

Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti

Sanjay Basu, Azadeh Aryan, Justin M. Overcash, Glady Hazitha Samuel, Michelle A. E. Anderson, Timothy J. Dahlem, Kevin M. Myles, and Zach N. Adelman
PNAS March 31, 2015 112 (13) 4038-4043; first published March 16, 2015; https://doi.org/10.1073/pnas.1502370112

  1. Edited by Anthony A. James, University of California, Irvine, CA, and approved February 24, 2015 (received for review February 6, 2015)

Article Figures & SI

Figures

  • Fig. 1.
    Fig. 1.

    Heterodimeric TALEN activity in mosquito embryos. (A) Representation of the TALEN constructs used to target Ae. aegypti kmo. All possessed 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 differed 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).

  • Fig. 2.
    Fig. 2.

    Somatic and germ-line CRISPR/Cas9 editing of Ae. aegypti. White-light photographs of WT eyes (A), G0 individuals showing somatic CRISPR-editing (B and C), and germ line-based CRISPR-edited in G1 progeny (D). (E) Analysis of indels in CRISPR-edited progeny. Top line represents WT sequence; subsequent lines show individual mutant sequences. Underlined text indicates the crRNA target sequence including the PAM (highlighted in blue) and cleavage point (red letters and black arrow); the kmo-exon5 TALEN spacer site is shown for comparison (gray). Deleted bases are denoted by dashes and inserted bases are shown in blue. The number of deleted (Δ) or inserted (+) bases and their occurrence are indicated to the right; in-frame mutations are indicated (*) to the left.

  • Fig. 3.
    Fig. 3.

    Suppression of NHEJ components increases recombination activity in mosquito embryos. Representation of the plasmid constructs used to detect SSA (A) or NHEJ (B). Relative light units observed from embryos 48 h after injection with the SSA sensor (C) or NHEJ sensor (D) in the presence of the indicated dsRNA. Each point represents a pool of ∼100 embryos. Data were trimmed by removing highest/lowest points from all samples (experimental and control). For C, statistical differences were determined by ANOVA followed by Dunnett's multiple comparison test; statistically different groups are indicated (*P < 0.05; **P < 0.01). For D, statistical differences were determined with a two-tailed t test (Mann-Whitney); P values are indicated.

  • Fig. 4.
    Fig. 4.

    Site-specific HDR-based gene insertion. (A) Representation of the HDR donor construct used to insert the PUb-EGFP expression cassette into exon5 of dcr2. Block arrows represent dcr2 exons 4–8; blue bars indicate sequences used as left (L) and right (R) homology arms in the circular donor construct. TALEN target site is indicated by red vertical line; PCR primers used to confirm integration into the target site are indicated by red arrowheads. (B) Transgenic larvae following site-specific insertion of the PUb-EGFP cassette. (C) PCR confirmation of HDR-mediated site-specific insertion of the PUb-EGFP transgene.

Tables

  • Table 1.

    Effectiveness of sgRNAs between and within genes

    Vectorbase IDGeneNo. sgRNAsHRMA+HMRA++ (>0.05)
    AAEL008879kmo875
    AAEL017365lig41021
    Noneku70431
    AAEL008687loqs12113
    AAEL011753r2d21061
    Nonenix842
    Total5233 (63%)13 (25%)
  • Table 2.

    CRISPR mutagenesis is highly efficient in Ae. aegypti

    TargetsgRNANo. injectedTotal G0Fertile G0Mutant G1†
    kmo519*830170 (20.5%)84 (49.4%)27/85 (32.1%)
    lig4#71,110176 (15.8%)ND8/23 (34.8%)
    ku70#3613172 (28%)77 (44.8%)10/42 (23.8%)
    r2d2#6565121 (21.4%)51 (42.1%)20/33 (60.6%)
    loqs#3548124 (22.6)62 (50%)27/30 (90.0%)

    • ND, not determined.

    • ↵† Number of G0 individuals that produced mutant progeny/total.

  • Table 3.

    Transgene insertion following suppression of NHEJ

    TargetHR armsdsRNANo. injectedNo. G0 (%)No. G0 poolsNo. G1 screenedEGFP+ (#)Rate†
    kmo0.6/0.5 kb—1,010109 (11%)6>11,0000<1.8%
    kmo2.2/1.7 kb—1,030152 (15%)8>12,0000<1.3%
    kmo2.2/1.7 kbegfp1,130192 (17%)9∼33,0000<1.0%
    kmo2.2/1.7 kbku70 #11,090166 (15%)9∼33,000P1 (1), P3 (36)2.4%
    kmo2.2/1.7 kbku70 #21,035118 (11%)5∼16,000P2 (8)1.7%
    dcr21/1.7 kbegfp1,138237 (21%)10∼46,0000<0.8%
    dcr21/1.7 kbku70 #21,050144 (14%)7∼23,000P1 (7), P2 (2)2.7%
    Total-/egfp3,298581 (18%)27∼91,0000<0.3%
    ku703,175428 (13%)21∼72,00052.3%

    • ↵† Minimum rate of gene knockin based on 50% fertility.

Data supplements

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CRISPR-based gene insertion in mosquitoes
Sanjay Basu, Azadeh Aryan, Justin M. Overcash, Glady Hazitha Samuel, Michelle A. E. Anderson, Timothy J. Dahlem, Kevin M. Myles, Zach N. Adelman
Proceedings of the National Academy of Sciences Mar 2015, 112 (13) 4038-4043; DOI: 10.1073/pnas.1502370112
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Proceedings of the National Academy of Sciences: 112 (13)
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