A CRISPR/Cas9 Vector System for Tissue-Specific Gene Disruption in Zebrafish

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Developmental Cell Supplemental Information A CRISPR/Cas9 Vector System for Tissue-Specific Gene Disruption in Zebrafish Julien Ablain, Ellen M. Durand, Song Yang, Yi Zhou, and Leonard I. Zon

% larvae per phenotype Supplemental Data A urod p53 Target sequences #1: AGTTCAGGGAATCACGGGCA #2: AGATCCTCAGGCTCCTTCAG GGTGGGAGAGTGGATGGCTG B grna p53 grna urod #2 - + - + 500 bp 500 bp C 100 WT Mild Intermediate Strong D Cas9 mrna + grna urod 80 * phenotype: WT Mild Int Strong 60 40 500 bp 20 0 Ø * urod wt mrna urod yq mrna Cas9 mrna + grna urod E Cas9 mrna + grna p53 Cas9 mrna + grna urod 50 hpf 300 µm Figure S1. CRISPR targeting of urod, but not p53, elicits a fluorescent phenotype in zebrafish embryos (related to Figure 1)

(A) CRISPR target sequences used to knockout the urod and p53 zebrafish genes. (B) T7E1 mutagenesis assay at the p53 CRISPR target site (left) and at the second urod site (right). The assay was performed on genomic DNA from 2 dpf embryos injected at the one-cell stage with Cas9 mrna and either the grna against p53 or the grna against urod. Arrows point to cleavage bands. (C) Quantification of the fluorescent phenotype obtained after co-injection of Cas9 mrna and a grna against urod ± wt urod mrna (urod wt ) or yquem urod mrna (urod mrna bearing the inactivating mutation found in the yquem mutant, urod yq ). WT: no fluorescent blood cell. Mild: <10 fluorescent blood cells. Intermediate: <50 fluorescent blood cells. Strong: >50 fluorescent blood cells. Data represented as mean percentage ± SD of 3 independent experiments. *: p<0.05, paired t-test. (D) T7E1 mutagenesis assay at the CRISPR target site in the urod gene. AB embryos were injected at the one-cell stage with Cas9 mrna and a grna against urod, and sorted into 4 groups (WT, mild, intermediate, high) according to their fluorescent phenotypes (see Figure S1C). The assay was performed on genomic DNA from 8 embryos from each group at 2 dpf. Arrows point to cleavage bands. (E) Confocal images at 50 hpf of embryos injected at the one-cell stage with Cas9 mrna and either the grna against p53 (negative control) or a grna against urod.

A GCGTCTTTTGTTCTGGTCATCAAGGAGGGGGGAATGTTCTGCGCATGCCTGTGGGGGAGGGAGAAGGA CACGTCACTGAAAACGTCCCTGCATCACACCGAGACACCCAATCACTCAAGCCGAGACCAGATAATTTT GCATATGCTTTACAGTTTGAAAAATACCACGGTAAACCCTCACACAAACTCTGGATTTGAGATCTTTCAGG TTTTATCAGTTTGCAGGTTTATGTCACCATGATATAGGGTCAGACTTGATCTAAGGGAGCTGAATAAGTG GTTTAGTCACTCACCACCTCCCAAAAACATACCCAGAAGTCCCTGGTATATATAGCTCTCCCTCCAGCTC TTGGTTCGCATATGACTCCTCAGTGACGAGGAGTCAGTAGCGTTTTAGAGCTAGAAATAGCAAGTTAAAA TAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT B U6-3 promoter BseRI sites grna scaffold uninjected Cas9 WISH ubi:cas9 48 hpf gata1:cas9 mylz2:cas9 C grna: urod p53 p53 urod E D Blood pcmlc2:gfp, U6:gRNA, gata1:cas9 p53 locus pcmlc2:gfp U6:gRNA urod urod locus D urod target locus AACAGAGTTCAGGGAATCACGGGCAGGGAAAGATT WT AACAGAGTTCAGGGAA AGATT -14 AACAGAGTTCAGGGAATCACGGGC del AACAGAGTTCAGGGAATCAC GGGAAAGATT -5 AACAGAGTTCAGGGAATCACGG CAGGGAAAGATT -1 AACAGAGTTCAGGGAATCACGGG GAAAGATT -4 AACAGAGTTCAGGGAATCA GGGAAAGATT -6 AACAGAGTTCAGGGAATCACGG AAAGATT -6 p53 target locus ATGTTGGTGGGAGAGTGGATGGCTGAGGCTGTTCT WT ctrl mylz2 gata1:cas9 ATGTTGGTGGGAGAGTGGATGGCTG del GCTGAGGCTGTTCT del CTGAGGCTGTTCT del ATGTTGGTGGGAGAGTGGATG AGGCTGTTCT -4 ATGTTGGTGGGAGAGTGGATGGCTG TTCT -6 Sorted muscle Figure S2. Tissue-specific expression of Cas9 and gene targeting with the CRISPR vector (related to Figure 2).

(A) Sequence of the portion of the vector presented in Figure 2A encompassing the U6 promoter and the grna scaffold. (B) Representative images of whole mount in situ hybridization (WISH) using an antisense RNA probe against Cas9 mrna in 48 hpf embryos injected with Tol2 mrna and the pcmlc2:gfp, U6:gRNA vectors expressing Cas9 under the control of the indicated promoters. (C) T7E1 mutagenesis assay at the CRISPR target sites in the p53 or urod genes. The assay was performed on genomic DNA from the blood of 2 dpf embryos injected at the one-cell stage with Tol2 mrna and the pcmlc2:gfp, U6:gRNA, gata1:cas9 vectors containing a grna against p53 or urod. Arrows point to cleavage bands. (D) Most frequent (>2%) mutant urod and p53 alleles found by deep-sequencing in the blood of embryos injected with Tol2 mrna and the pcmlc2:gfp, U6:gRNA, gata1:cas9 vectors containing grnas against urod and p53 respectively. The CRISPR target sequence is in green, mutations in red. The type of mutation is indicated (del: large deletion). (E) T7E1 mutagenesis assay at the CRISPR target site in the urod gene. The assay was performed on genomic DNA from sorted mcherry-positive cells from 2 dpf Tg(mylz2:mCherry) embryos injected at the one-cell stage with Tol2 mrna and the pcmlc2:gfp, U6:gRNA urod, mylz2:cas9 or pcmlc2:gfp, U6:gRNA urod, gata1:cas9 vectors.

% larvae per phenotype A pcmlc2:gfp, U6:gRNA urod, ubi:cas9 pcmlc2:gfp, U6:gRNA urod, gata1:cas9 50 hpf pcmlc2:gfp, U6:gRNA urod, mylz2:cas9 pcmlc2:gfp, U6:gRNA p53, gata1:cas9 300 µm B 100 WT Mild Intermediate Strong 80 60 40 20 0 ubi:cas9 gata1:cas9 mylz2:cas9 pcmlc2:gfp, U6:gRNA urod C D pa2 U6:gRNA urod gata1:cas9- T2A-GFP Figure S3. Fluorescent phenotype induced by CRISPR vectors targeting urod (related to Figure 3).

(A) Confocal images of 50 hpf embryos injected with Tol2 mrna and pcmlc2:gfp, U6:gRNA urod vectors expressing Cas9 under the control of the indicated promoters. (B) Quantification of the fluorescent phenotype presented in Figure 3A. WT: no fluorescent blood cell. Mild: <10 fluorescent blood cells. Intermediate: <50 fluorescent blood cells. Strong: >50 fluorescent blood cells. Ubi:Cas9 (n=61), gata1:cas9 (n=51), mylz2:cas9 (n=43). (C) Schematic representation of a variant tissue-specific CRISPR vector. Gata1 promoter (Pgata1) drives both Cas9 and GFP expression through a self-cleaving T2A peptide. Any grna of interest can be produced ubiquitously off the U6 promoter. Tol2 indicates transposition sites for the Tol2 transposase. pa: SV40 polya sequence. (D) Confocal images of the yolk region of 30 hpf embryos injected with Tol2 mrna and the pa2, U6:gRNA urod, gata1:cas9-t2a-gfp vector. Arrows point to cells that are both green and red. Scale bar: 20 µm. Note the small size of red-only cells.

Relative Cas9 expression (ΔΔCt) pcmlc2:gfp U6:gRNA urod gata1:cas9 % larvae per phenotype WT siblings A pcmlc2:gfp, U6:gRNA urod, gata1:cas9 F1 pcmlc2:gfp, U6:gRNA urod, mylz2:cas9 50 hpf 300 µm B F1, 36 hpf C Blood GFP in the heart - + E 100 80 60 D 14 mutant alleles out of 20 sequenced alleles CAGAGTTCAGGGAATCACGGGCA GGGAAAGATT WT x6 CAGAGTTCAGGGAATCACGG CAGGGAAAGATT -1 x4 CAGAGTTCAGGGAATCAC GGGAAAGATT -5 x2 CAGAGTTCAG CAGGGAAAGATT -11 x2 CAGAGTTCAGGGAA AGATT -14 CAGAGTTCAGGGAATCAC AGGGAAAGATT -4 CAGAGTTCAGGGAATCACGGGC del CAGAGTTCAGGGAATCACG CAGGGAAAGATT -2 CAGAGTTCAGGGAATCACGG AAAGATT -6 CAGAGTTCAGGGAAT A GAAAGATT -10;+1 F 40 20 0 2,5 2 1,5 1 0,5 0-0,5 ubi:cas9 gata1:cas9 Mild Full p = 0.004 Mild Full Figure S4. Analysis of the urod phenotype in stable F1 embryos (related to Figure 4).

(A) Confocal images of 50 hpf embryos from the F1 generation of fish stably expressing the indicated vectors. (B) FACS analysis of the blood of 36 hpf F1 embryos stably expressing the pcmlc2:gfp, U6:gRNA urod, gata1:cas9 vector (bottom panels). Blood from F1 siblings not presenting green hearts was used as a negative control (top panels). Red fluorescence due to urod inactivation was detected in the PE-Cy5 channel (right panels). Cytox blue was used to mark dead or dying cells (left panels). (C) T7E1 mutagenesis assay at the CRISPR target site in the urod gene. The assay was performed on genomic DNA from the blood of 2 dpf F1 embryos stably expressing the pcmlc2:gfp, U6:gRNA urod, gata1:cas9 vector. Blood from F1 siblings not presenting green hearts was used as a negative control. (D) Sequences of the mutant urod alleles found in the blood of embryos stably expressing the pcmlc2:gfp, U6:gRNA urod, gata1:cas9 vector. The CRISPR target sequence is in green, mutations in red. The type of mutation and the associated number of detected alleles are indicated. (E) Quantification of the fluorescent phenotype observed in F1 embryos. Mild: <10 fluorescent blood cells. Full denotes a number of fluorescent cells comparable to that observed in age-matched LCR:GFP embryos. Ubi:Cas9 (n=37), gata1:cas9 (n=80). (F) qpcr analysis of Cas9 expression in F1 embryos stably expressing pcmlc2:gfp, U6:gRNA urod, ubi:cas9 vector. Four embryos showing no or few fluorescent cells (mild) and four embryos showing a full phenotype were compared. Data represented as mean ± SD.

Mutation index F0 ubi promoter (whole embryos) F0 gata1 promoter (blood) F1 ubi promoter (whole embryos) F1 gata1 promoter (blood) Deep-sequencing 36% 25% NA NA TA cloning 40% 33% 82% 70% Table S1. Mutation indexes for the pcmlc2:gfp, U6:gRNA urod, ubi:cas9 and pcmlc2:gfp, U6:gRNA urod, gata1:cas9 vectors (related to Figure 4). The index was calculated as the number of mutated alleles over the total number of sequenced alleles using either of two methods: deep-sequencing or TA cloning followed by Sanger sequencing of PCR amplicons around the CRISPR target site in the urod gene. Mutation indexes for both injected F0 and stable F1 embryos are indicated. Movie S1. Circulating fluorescent erythrocytes after tissue-specific urod targeting (related to Figure 3). Live confocal imaging of a 50 hpf embryo injected with Tol2 mrna and the pcmlc2:gfp, U6:gRNA urod, gata1:cas9 vector at the one-cell stage.

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