Student or educator? Sign up with your school email and get 6 months of Pro free.
CoChat
Sign Up
Cited by 32CRISPR and Genetic EngineeringZebrafish Biomedical Research ApplicationsDevelopmental Biology and Gene Regulation

The DOI matches its Crossref record; 65 of the 65 references it cites were found in a registry or an index.

Highly Efficient Synthetic CRISPR RNA/Cas9-Based Mutagenesis for Rapid Cardiovascular Phenotypic Screening in F0 Zebrafish

Open access
Quick, Rachael E., Buck, Luke D., Parab, Sweta, Tolbert, Zane R., Matsuoka, Ryota L.·2021·Frontiers in Cell and Developmental Biology
DOI 10.3389/fcell.2021.735598PMC PMC8570140

Published version of a preprint

Open this paper in CoChat — free account

Abstract

The zebrafish is a valuable vertebrate model to study cardiovascular formation and function due to the facile visualization and rapid development of the circulatory system in its externally growing embryos. Despite having distinct advantages, zebrafish have paralogs of many important genes, making reverse genetics approaches inefficient since generating animals bearing multiple gene mutations requires substantial efforts. Here, we present a simple and robust synthetic CRISPR RNA/Cas9-based mutagenesis approach for generating biallelic F0 zebrafish knockouts. Using a dual-guide synthetic CRISPR RNA/Cas9 ribonucleoprotein (dgRNP) system, we compared the efficiency of biallelic gene disruptions following the injections of one, two, and three dgRNPs per gene into the cytoplasm or yolk. We show that simultaneous cytoplasmic injections of three distinct dgRNPs per gene into one-cell stage embryos resulted in the most efficient and consistent biallelic gene disruptions. Importantly, this triple dgRNP approach enables efficient inactivation of cell autonomous and cell non-autonomous gene function, likely due to the low mosaicism of biallelic disruptions. In support of this finding, we provide evidence that the F0 animals generated by this method fully phenocopied the endothelial and peri-vascular defects observed in corresponding stable mutant homozygotes. Moreover, this approach faithfully recapitulated the trunk vessel phenotypes resulting from the genetic interaction between two vegfr2 zebrafish paralogs. Mechanistically, investigation of genome editing and mRNA decay indicates that the combined mutagenic actions of three dgRNPs per gene lead to an increased probability of frameshift mutations, enabling efficient biallelic gene disruptions. Therefore, our approach offers a highly robust genetic platform to quickly assess novel and redundant gene function in F0 zebrafish.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.3389/fcell.2021.735598

Cites

65 of 65 references verified0 citation errors · 0 not checkable

Statistics

Figures

FIGURE 1 Embryos injected with the triple dgRNPs targeting kdrl exhibited aISV defects similar to those observed in kdrl–/– fish. (A) Experimental setup for the phenotypic analysis presented in panels (B–H). Adult kdrl+ /– fish were incrossed, and progeny generated from the crosses were imaged to quantify the lengths of their arterial intersegmental vessels (aISVs) at 32 hpf. (B–G) Lateral views of kdrl+/+ (B,E), kdrl+ /– (C,F), and kdrl–/– (D,G) trunk vasculature visualized by Tg(kdrl:EGFP) expression at 32 (B–D) and 55 (E–G) hpf. (H) Quantification of aISV lengths in kdrl+/+, kdrl+ /–, and kdrl–/– embryos at 32 hpf (n = 23 for kdrl+/+, n = 45 for kdrl+ /–, and n = 21 for kdrl–/– fish). Lengths of 5 aISVs per embryo were measured. NS: not significant. (I) Three synthetic CRISPR RNAs (crRNAs) were designed to target sequences within exon 2 (E2), E3, and E5 on the kdrl genomic locus. (J) Predicted domain structure of zebrafish Kdrl. Kdrl consists of a signal peptide (SP), seven immunoglobulin-like domains (Ig), a transmembrane domain (TM), and two tyrosine kinase domains (TyrKc). Arrows indicate the approximate positions of the protein sequences corresponding to the target sequences of the three designed crRNAs. (K–M) High-resolution melt analysis (HRMA) used to validate the efficacy of the three designed crRNAs. The melting curves of 6 independent embryos injected with the dgRNP complex containing kdrl crRNA1 (K, pink), crRNA2 (L, orange), or crRNA3 (M, purple) are presented. The melting curves of 2 independent, uninjected (K–M, blue) and Cas9 protein-injected (K–M, green) sibling embryos are also presented for comparison for each crRNA. Injection of each dgRNP cocktail disrupted the corresponding target genomic sequences. (N) Experimental workflow of the microinjection experiments for panels (O–U). Injection cocktails containing Cas9 protein with the individual or the three kdrl crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage Tg(kdrl:EGFP) embryos. Injected Tg(kdrl:EGFP) progeny were analyzed for aISV formation at 32 and 55 hpf. (O,P) Brightfield images of the 32 hpf embryos injected with Cas9 protein with (P) or without (O) the three kdrl crRNA:tracrRNA duplexes (crRNA1-3). (Q–T) Lateral trunk views of the 32 (Q,R) and 55 (S,T) hpf Tg(kdrl:EGFP) fish injected with Cas9 protein with (R,T) or without (Q,S) the three kdrl crRNA1-3. While aISVs in Cas9-injected embryos fused at the dorsal side of the trunk to form the dorsal longitudinal anastomotic vessel by 32 hpf (Q), a vast majority of aISVs in the embryos injected with the triple kdrl dgRNPs failed to reach the dorsal side of the trunk (R). This aISV stalling phenotype was observed at later developmental stages, including at 55 hpf (T). (U) Quantification of aISV lengths of 32 hpf embryos of indicated treatment (n = 20 fish for each treatment group; 5 aISV lengths per embryo measured). Average aISV lengths of the triple dgRNPs-injected embryos closely resembled those of kdrl–/– fish. A one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to calculate P values for panels (H,U). Scale bars: 50 μm in (D,G,R,T); 500 μm in (P).
FIGURE 1 Embryos injected with the triple dgRNPs targeting kdrl exhibited aISV defects similar to those observed in kdrl–/– fish. (A) Experimental setup for the phenotypic analysis presented in panels (B–H). Adult kdrl+ /– fish were incrossed, and progeny generated from the crosses were imaged to quantify the lengths of their arterial intersegmental vessels (aISVs) at 32 hpf. (B–G) Lateral views of kdrl+/+ (B,E), kdrl+ /– (C,F), and kdrl–/– (D,G) trunk vasculature visualized by Tg(kdrl:EGFP) expression at 32 (B–D) and 55 (E–G) hpf. (H) Quantification of aISV lengths in kdrl+/+, kdrl+ /–, and kdrl–/– embryos at 32 hpf (n = 23 for kdrl+/+, n = 45 for kdrl+ /–, and n = 21 for kdrl–/– fish). Lengths of 5 aISVs per embryo were measured. NS: not significant. (I) Three synthetic CRISPR RNAs (crRNAs) were designed to target sequences within exon 2 (E2), E3, and E5 on the kdrl genomic locus. (J) Predicted domain structure of zebrafish Kdrl. Kdrl consists of a signal peptide (SP), seven immunoglobulin-like domains (Ig), a transmembrane domain (TM), and two tyrosine kinase domains (TyrKc). Arrows indicate the approximate positions of the protein sequences corresponding to the target sequences of the three designed crRNAs. (K–M) High-resolution melt analysis (HRMA) used to validate the efficacy of the three designed crRNAs. The melting curves of 6 independent embryos injected with the dgRNP complex containing kdrl crRNA1 (K, pink), crRNA2 (L, orange), or crRNA3 (M, purple) are presented. The melting curves of 2 independent, uninjected (K–M, blue) and Cas9 protein-injected (K–M, green) sibling embryos are also presented for comparison for each crRNA. Injection of each dgRNP cocktail disrupted the corresponding target genomic sequences. (N) Experimental workflow of the microinjection experiments for panels (O–U). Injection cocktails containing Cas9 protein with the individual or the three kdrl crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage Tg(kdrl:EGFP) embryos. Injected Tg(kdrl:EGFP) progeny were analyzed for aISV formation at 32 and 55 hpf. (O,P) Brightfield images of the 32 hpf embryos injected with Cas9 protein with (P) or without (O) the three kdrl crRNA:tracrRNA duplexes (crRNA1-3). (Q–T) Lateral trunk views of the 32 (Q,R) and 55 (S,T) hpf Tg(kdrl:EGFP) fish injected with Cas9 protein with (R,T) or without (Q,S) the three kdrl crRNA1-3. While aISVs in Cas9-injected embryos fused at the dorsal side of the trunk to form the dorsal longitudinal anastomotic vessel by 32 hpf (Q), a vast majority of aISVs in the embryos injected with the triple kdrl dgRNPs failed to reach the dorsal side of the trunk (R). This aISV stalling phenotype was observed at later developmental stages, including at 55 hpf (T). (U) Quantification of aISV lengths of 32 hpf embryos of indicated treatment (n = 20 fish for each treatment group; 5 aISV lengths per embryo measured). Average aISV lengths of the triple dgRNPs-injected embryos closely resembled those of kdrl–/– fish. A one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to calculate P values for panels (H,U). Scale bars: 50 μm in (D,G,R,T); 500 μm in (P).CC BY · PubMed Central
FIGURE 2 Larvae injected with three unique flt4 dgRNPs phenocopied the complete loss of brain meningeal perivascular FGPs observed in flt4–/– fish. (A) Schematic representation of the dorsal view of the zebrafish larval head. The boxed area indicates the approximate region where the confocal images of panels (B–D) were captured. (B–D) Dorsal head views of 6 dpf flt4+/+, flt4+/–, and flt4–/– larvae carrying Tg(fli1:nEGFP) and Tg(lyve1:DsRed) transgenes. While flt4+/+ and flt4+/– larvae formed Tg(fli1:nEGFP);Tg(lyve1:DsRed)-double positive FGPs in the dorsal meningeal surfaces over the optic tectum (arrows, B,C), flt4–/– larvae completely lacked FGPs (D). (E) Quantification of FGPs over the optic tectum for each genotype at 6 dpf (n = 17 for flt4+/+, n = 39 for flt4+/–, and n = 21 for flt4–/– fish). (F) Three synthetic crRNAs were designed to target sequences within exon 3 (E3) and E6 on the flt4 genomic locus. (G) Predicted domain structure of zebrafish Flt4. Flt4 consists of a signal peptide (SP), six immunoglobulin-like domains (Ig), a transmembrane domain (TM), and two tyrosine kinase domains (TyrKc). Arrows indicate the approximate positions of the protein sequences corresponding to the target sequences of the three designed crRNAs. (H–J) HRMA used to validate the efficacy of the three designed crRNAs. The melting curves of 6 independent embryos injected with the dgRNP complex containing flt4 crRNA1 (H, pink), crRNA2 (I, orange), or crRNA3 (J, purple) are presented. The melting curves of 2 independent, uninjected (H–J, blue) and Cas9-injected (H–J, green) sibling embryos are also presented for comparison for each crRNA. Injection of each dgRNP cocktail disrupted the corresponding target genomic sequences. (K) Experimental workflow of the microinjection experiments for panels (L–R). Injection cocktails containing Cas9 protein with and without the individual or the three flt4 crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage Tg(fli1:nEGFP);Tg(lyve1:DsRed) embryos. Injected progeny were analyzed at 6 and 10 dpf for FGP formation over the optic tectum. (L,M) Brightfield images of the 6 dpf larvae injected with Cas9 protein with (M) or without (L) the three flt4 crRNA:tracrRNA duplexes (crRNA1-3). (N–Q) Dorsal head views of the 6 (N,O) and 10 (P,Q) dpf Tg(fli1:nEGFP);Tg(lyve1:DsRed) larvae injected with Cas9 protein with (O,Q) or without (N,P) the three flt4 crRNA1-3. While larvae injected with Cas9 alone formed FGPs over the optic tectum at 6 and 10 dpf (arrows, N,P), those injected with the three flt4 dgRNPs displayed a complete lack of the FGPs at both stages (O,Q). (R) Quantification of FGPs over the optic tectum at 6 dpf (n = 28 for uninjected; n = 24 for Cas9 controls; n = 32 for crRNA1, crRNA2, and crRNA3; and n = 35 for crRNA1-3). Larvae injected with all three flt4 dgRNPs failed to form FGPs. Fish injected with the individual flt4 dgRNPs displayed varying degrees of FGP formation. A one-way ANOVA followed by Tukey’s post hoc test was used to calculate P values for panels (E,R). Scale bars: 50 μm in (D,O,Q); 1 mm in (M).
FIGURE 2 Larvae injected with three unique flt4 dgRNPs phenocopied the complete loss of brain meningeal perivascular FGPs observed in flt4–/– fish. (A) Schematic representation of the dorsal view of the zebrafish larval head. The boxed area indicates the approximate region where the confocal images of panels (B–D) were captured. (B–D) Dorsal head views of 6 dpf flt4+/+, flt4+/–, and flt4–/– larvae carrying Tg(fli1:nEGFP) and Tg(lyve1:DsRed) transgenes. While flt4+/+ and flt4+/– larvae formed Tg(fli1:nEGFP);Tg(lyve1:DsRed)-double positive FGPs in the dorsal meningeal surfaces over the optic tectum (arrows, B,C), flt4–/– larvae completely lacked FGPs (D). (E) Quantification of FGPs over the optic tectum for each genotype at 6 dpf (n = 17 for flt4+/+, n = 39 for flt4+/–, and n = 21 for flt4–/– fish). (F) Three synthetic crRNAs were designed to target sequences within exon 3 (E3) and E6 on the flt4 genomic locus. (G) Predicted domain structure of zebrafish Flt4. Flt4 consists of a signal peptide (SP), six immunoglobulin-like domains (Ig), a transmembrane domain (TM), and two tyrosine kinase domains (TyrKc). Arrows indicate the approximate positions of the protein sequences corresponding to the target sequences of the three designed crRNAs. (H–J) HRMA used to validate the efficacy of the three designed crRNAs. The melting curves of 6 independent embryos injected with the dgRNP complex containing flt4 crRNA1 (H, pink), crRNA2 (I, orange), or crRNA3 (J, purple) are presented. The melting curves of 2 independent, uninjected (H–J, blue) and Cas9-injected (H–J, green) sibling embryos are also presented for comparison for each crRNA. Injection of each dgRNP cocktail disrupted the corresponding target genomic sequences. (K) Experimental workflow of the microinjection experiments for panels (L–R). Injection cocktails containing Cas9 protein with and without the individual or the three flt4 crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage Tg(fli1:nEGFP);Tg(lyve1:DsRed) embryos. Injected progeny were analyzed at 6 and 10 dpf for FGP formation over the optic tectum. (L,M) Brightfield images of the 6 dpf larvae injected with Cas9 protein with (M) or without (L) the three flt4 crRNA:tracrRNA duplexes (crRNA1-3). (N–Q) Dorsal head views of the 6 (N,O) and 10 (P,Q) dpf Tg(fli1:nEGFP);Tg(lyve1:DsRed) larvae injected with Cas9 protein with (O,Q) or without (N,P) the three flt4 crRNA1-3. While larvae injected with Cas9 alone formed FGPs over the optic tectum at 6 and 10 dpf (arrows, N,P), those injected with the three flt4 dgRNPs displayed a complete lack of the FGPs at both stages (O,Q). (R) Quantification of FGPs over the optic tectum at 6 dpf (n = 28 for uninjected; n = 24 for Cas9 controls; n = 32 for crRNA1, crRNA2, and crRNA3; and n = 35 for crRNA1-3). Larvae injected with all three flt4 dgRNPs failed to form FGPs. Fish injected with the individual flt4 dgRNPs displayed varying degrees of FGP formation. A one-way ANOVA followed by Tukey’s post hoc test was used to calculate P values for panels (E,R). Scale bars: 50 μm in (D,O,Q); 1 mm in (M).CC BY · PubMed Central
FIGURE 3 Larvae injected with three unique ccbe1 dgRNPs phenocopied the complete loss of brain meningeal perivascular FGPs observed in ccbe1–/– fish. (A) Schematic representation of the dorsal view of the zebrafish larval head. The boxed area indicates the approximate region where the confocal images of panels (B–D) were captured. (B–D) Dorsal head views of 6 dpf ccbe1+/+, ccbe1+/–, and ccbe1–/– larvae carrying Tg(fli1:nEGFP) and Tg(lyve1:DsRed) transgenes. While ccbe1+/+ and ccbe1+/– larvae formed Tg(fli1:nEGFP);Tg(lyve1:DsRed)-double positive FGPs in the dorsal meningeal surfaces over the optic tectum (arrows, B,C), ccbe1–/– larvae completely lacked FGPs (D). (E) Quantification of FGPs over the optic tectum for each genotype at 6 dpf (n = 16 for ccbe1+/+, n = 22 for ccbe1+/–, and n = 15 for ccbe1–/– fish). (F) Three synthetic crRNAs were designed to target sequences within exon 1 (E1), E2, and E3 on the ccbe1 genomic locus. (G) Predicted domain structure of zebrafish Ccbe1. Ccbe1 consists of a signal peptide (SP), an EGF domain (EGF), a calcium-binding EGF domain (Ca-EGF), and two collagen repeat domains (ColA and ColB). Arrows indicate the approximate positions of the protein sequences corresponding to the target sequences of the three designed crRNAs. (H–J) HRMA used to validate the efficacy of the three designed crRNAs. The melting curves of 6 independent embryos injected with the dgRNP complex containing ccbe1 crRNA1 (H, pink), crRNA2 (I, orange), or crRNA3 (J, purple) are presented. The melting curves of 2 independent, uninjected (H–J, blue) and Cas9-injected (H–J, green) sibling embryos are also presented for comparison for each crRNA. Injection of each dgRNP cocktail disrupted the corresponding target genomic sequences. (K) Experimental workflow of the microinjection experiments for panels (L–R). Injection cocktails containing Cas9 protein with and without the individual or the three ccbe1 crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage Tg(fli1:nEGFP);Tg(lyve1:DsRed) embryos. Injected progeny were analyzed at 6 and 10 dpf for FGP formation over the optic tectum. (L,M) Brightfield images of the 6 dpf larvae injected with Cas9 protein with (M) or without (L) the three ccbe1 crRNA:tracrRNA duplexes (crRNA1-3). (N–Q) Dorsal head views of the 6 (N,O) and 10 (P,Q) dpf Tg(fli1:nEGFP);Tg(lyve1:DsRed) larvae injected with Cas9 protein with (O,Q) or without (N,P) the three ccbe1 crRNA1-3. Larvae injected with Cas9 alone formed FGPs over the optic tectum at 6 and 10 dpf (arrows, N,P). However, fish injected with the three ccbe1 dgRNPs completely lacked the FGPs at both stages (O,Q). (R) Quantification of FGPs over the optic tectum at 6 dpf (n = 24 for uninjected and Cas9 controls; n = 30 for crRNA1, crRNA2, and crRNA3; and n = 35 for crRNA1-3). All larvae injected with the three ccbe1 dgRNPs exhibited a complete loss of FGPs over the optic tectum. Fish injected with the individual ccbe1 dgRNPs displayed varying degrees of FGP formation. A one-way ANOVA followed by Tukey’s post hoc test was used to calculate P values for panels (E,R). Scale bars: 50 μm in (D,O,Q); 1 mm in (M).
FIGURE 3 Larvae injected with three unique ccbe1 dgRNPs phenocopied the complete loss of brain meningeal perivascular FGPs observed in ccbe1–/– fish. (A) Schematic representation of the dorsal view of the zebrafish larval head. The boxed area indicates the approximate region where the confocal images of panels (B–D) were captured. (B–D) Dorsal head views of 6 dpf ccbe1+/+, ccbe1+/–, and ccbe1–/– larvae carrying Tg(fli1:nEGFP) and Tg(lyve1:DsRed) transgenes. While ccbe1+/+ and ccbe1+/– larvae formed Tg(fli1:nEGFP);Tg(lyve1:DsRed)-double positive FGPs in the dorsal meningeal surfaces over the optic tectum (arrows, B,C), ccbe1–/– larvae completely lacked FGPs (D). (E) Quantification of FGPs over the optic tectum for each genotype at 6 dpf (n = 16 for ccbe1+/+, n = 22 for ccbe1+/–, and n = 15 for ccbe1–/– fish). (F) Three synthetic crRNAs were designed to target sequences within exon 1 (E1), E2, and E3 on the ccbe1 genomic locus. (G) Predicted domain structure of zebrafish Ccbe1. Ccbe1 consists of a signal peptide (SP), an EGF domain (EGF), a calcium-binding EGF domain (Ca-EGF), and two collagen repeat domains (ColA and ColB). Arrows indicate the approximate positions of the protein sequences corresponding to the target sequences of the three designed crRNAs. (H–J) HRMA used to validate the efficacy of the three designed crRNAs. The melting curves of 6 independent embryos injected with the dgRNP complex containing ccbe1 crRNA1 (H, pink), crRNA2 (I, orange), or crRNA3 (J, purple) are presented. The melting curves of 2 independent, uninjected (H–J, blue) and Cas9-injected (H–J, green) sibling embryos are also presented for comparison for each crRNA. Injection of each dgRNP cocktail disrupted the corresponding target genomic sequences. (K) Experimental workflow of the microinjection experiments for panels (L–R). Injection cocktails containing Cas9 protein with and without the individual or the three ccbe1 crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage Tg(fli1:nEGFP);Tg(lyve1:DsRed) embryos. Injected progeny were analyzed at 6 and 10 dpf for FGP formation over the optic tectum. (L,M) Brightfield images of the 6 dpf larvae injected with Cas9 protein with (M) or without (L) the three ccbe1 crRNA:tracrRNA duplexes (crRNA1-3). (N–Q) Dorsal head views of the 6 (N,O) and 10 (P,Q) dpf Tg(fli1:nEGFP);Tg(lyve1:DsRed) larvae injected with Cas9 protein with (O,Q) or without (N,P) the three ccbe1 crRNA1-3. Larvae injected with Cas9 alone formed FGPs over the optic tectum at 6 and 10 dpf (arrows, N,P). However, fish injected with the three ccbe1 dgRNPs completely lacked the FGPs at both stages (O,Q). (R) Quantification of FGPs over the optic tectum at 6 dpf (n = 24 for uninjected and Cas9 controls; n = 30 for crRNA1, crRNA2, and crRNA3; and n = 35 for crRNA1-3). All larvae injected with the three ccbe1 dgRNPs exhibited a complete loss of FGPs over the optic tectum. Fish injected with the individual ccbe1 dgRNPs displayed varying degrees of FGP formation. A one-way ANOVA followed by Tukey’s post hoc test was used to calculate P values for panels (E,R). Scale bars: 50 μm in (D,O,Q); 1 mm in (M).CC BY · PubMed Central
FIGURE 4 Larvae injected with three unique vegfab dgRNPsphenocopied the complete loss of VTAs observed in vegfab–/– fish. (A) Schematic representation of the lateral view of an 8 dpf zebrafish larva. The boxed area indicates the approximate trunk region where all VTA quantifications were performed. (B–D) Lateral trunk views of 8 dpf vegfab+/+, vegfab+/–, and vegfab–/– larvae carrying Tg(kdrl:EGFP) and Tg(kdrl:NLS-mCherry) transgenes. While vegfab+/+ larvae formed the VTAs, which extended bilaterally along the ventrolateral sides of the spinal cord (B, arrows), vegfab–/– larvae formed no VTAs (D). Most vegfab+/– larvae exhibited partially forming VTAs at this stage (C, arrows). (E) Quantification of endothelial cells (ECs) at the VTAs within a 5 somite region per animal at 8 dpf (n = 27 for vegfab+/+, n = 64 for vegfab+/–, and n = 35 for vegfab–/– fish). (F) Three synthetic crRNAs were designed to target sequences within exon 1 (E1), E2, and E3 on the vegfab genomic locus. (G) Predicted domain structure of zebrafish Vegfab171 isoform. Vegfab171 consists of a signal peptide (SP), a VEGF homology domain (VHD), and a heparin and neuropilin1 binding domain (H/N). Arrows indicate the approximate positions of the protein sequences corresponding to the target sequences of the three designed crRNAs. (H–J) HRMA used to validate the efficacy of the three designed crRNAs. The melting curves of 6 independent embryos injected with the dgRNP complex containing vegfab crRNA1 (H, pink), crRNA2 (I, orange), or crRNA3 (J, purple) are presented. The melting curves of 2 independent, uninjected (H–J, blue) and Cas9-injected (H–J, green) sibling embryos are also presented for comparison for each crRNA. Injection of each dgRNP cocktail disrupted the corresponding target genomic sequences. (K) Experimental workflow of the microinjection experiments for panels (L–R). Injection cocktails containing Cas9 protein with and without the individual or the three vegfab crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage Tg(kdrl:EGFP);Tg(kdrl:NLS-mCherry) embryos. Injected progeny were analyzed for VTA formation at 8 and 12 dpf. (L,M) Brightfield images of the 8 dpf larvae injected with Cas9 protein with (M) or without (L) the three vegfab crRNA:tracrRNA duplexes (crRNA1-3). (N–Q) Lateral trunk views of the 8 (N,O) and 12 (P,Q) dpf Tg(kdrl:EGFP);Tg(kdrl:NLS-mCherry) larvae injected with Cas9 protein with (O,Q) or without (N,P) the three vegfab crRNA1-3. While larvae injected with Cas9 alone formed most of the VTAs at 8 and 12 dpf (arrows, N,P), those injected with the three vegfab dgRNPs completely lacked VTAs at both stages (O,Q). (R) Quantification of ECs at the VTAs within a 5 somite region per animal at 8 dpf (n = 25 for uninjected and Cas9 controls; n = 30 for crRNA1, crRNA2, and crRNA3; and n = 32 for crRNA1-3). All larvae injected with the three vegfab dgRNPs failed to form VTAs. Fish injected with the individual vegfab dgRNPs displayed varying degrees of VTA formation. A one-way ANOVA followed by Tukey’s post hoc test was used to calculate P values for panels (E,R). Scale bars: 50 μm in (D,O,Q); 1 mm in (M).
FIGURE 4 Larvae injected with three unique vegfab dgRNPsphenocopied the complete loss of VTAs observed in vegfab–/– fish. (A) Schematic representation of the lateral view of an 8 dpf zebrafish larva. The boxed area indicates the approximate trunk region where all VTA quantifications were performed. (B–D) Lateral trunk views of 8 dpf vegfab+/+, vegfab+/–, and vegfab–/– larvae carrying Tg(kdrl:EGFP) and Tg(kdrl:NLS-mCherry) transgenes. While vegfab+/+ larvae formed the VTAs, which extended bilaterally along the ventrolateral sides of the spinal cord (B, arrows), vegfab–/– larvae formed no VTAs (D). Most vegfab+/– larvae exhibited partially forming VTAs at this stage (C, arrows). (E) Quantification of endothelial cells (ECs) at the VTAs within a 5 somite region per animal at 8 dpf (n = 27 for vegfab+/+, n = 64 for vegfab+/–, and n = 35 for vegfab–/– fish). (F) Three synthetic crRNAs were designed to target sequences within exon 1 (E1), E2, and E3 on the vegfab genomic locus. (G) Predicted domain structure of zebrafish Vegfab171 isoform. Vegfab171 consists of a signal peptide (SP), a VEGF homology domain (VHD), and a heparin and neuropilin1 binding domain (H/N). Arrows indicate the approximate positions of the protein sequences corresponding to the target sequences of the three designed crRNAs. (H–J) HRMA used to validate the efficacy of the three designed crRNAs. The melting curves of 6 independent embryos injected with the dgRNP complex containing vegfab crRNA1 (H, pink), crRNA2 (I, orange), or crRNA3 (J, purple) are presented. The melting curves of 2 independent, uninjected (H–J, blue) and Cas9-injected (H–J, green) sibling embryos are also presented for comparison for each crRNA. Injection of each dgRNP cocktail disrupted the corresponding target genomic sequences. (K) Experimental workflow of the microinjection experiments for panels (L–R). Injection cocktails containing Cas9 protein with and without the individual or the three vegfab crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage Tg(kdrl:EGFP);Tg(kdrl:NLS-mCherry) embryos. Injected progeny were analyzed for VTA formation at 8 and 12 dpf. (L,M) Brightfield images of the 8 dpf larvae injected with Cas9 protein with (M) or without (L) the three vegfab crRNA:tracrRNA duplexes (crRNA1-3). (N–Q) Lateral trunk views of the 8 (N,O) and 12 (P,Q) dpf Tg(kdrl:EGFP);Tg(kdrl:NLS-mCherry) larvae injected with Cas9 protein with (O,Q) or without (N,P) the three vegfab crRNA1-3. While larvae injected with Cas9 alone formed most of the VTAs at 8 and 12 dpf (arrows, N,P), those injected with the three vegfab dgRNPs completely lacked VTAs at both stages (O,Q). (R) Quantification of ECs at the VTAs within a 5 somite region per animal at 8 dpf (n = 25 for uninjected and Cas9 controls; n = 30 for crRNA1, crRNA2, and crRNA3; and n = 32 for crRNA1-3). All larvae injected with the three vegfab dgRNPs failed to form VTAs. Fish injected with the individual vegfab dgRNPs displayed varying degrees of VTA formation. A one-way ANOVA followed by Tukey’s post hoc test was used to calculate P values for panels (E,R). Scale bars: 50 μm in (D,O,Q); 1 mm in (M).CC BY · PubMed Central
FIGURE 5 Yolk injections of three dgRNPs per gene allowed efficient generation of biallelic F0 knockouts but were less consistent than cytoplasmic injections. (A) Experimental workflow of the yolk microinjection experiments for panel (B). Injection cocktails containing Cas9 protein with and without the three flt4 or ccbe1 crRNA:tracrRNA duplexes were injected into the yolk of one-cell stage Tg(fli1:nEGFP);Tg(lyve1:DsRed) embryos. Injected progeny were analyzed at 6 dpf for FGP formation over the optic tectum. (B) Quantification of FGPs over the optic tectum at 6 dpf after yolk injection of Cas9 protein with and without the three flt4 or ccbe1 crRNA:tracrRNA duplexes (n = 20 for Cas9 controls, n = 35 for flt4 crRNA1-3 and ccbe1 crRNA1-3). All flt4 dgRNPs-injected larvae except one failed to form FGPs over the optic tectum. Approximately 89% of ccbe1 dgRNPs-injected larvae completely lacked FGPs, but the remaining fish formed FGPs in a manner comparable to Cas9-injected controls. A one-way ANOVA followed by Dunnett’s post hoc test was used to calculate P values. (C) Percentage of 6 dpf larvae of indicated treatment with and without FGPs over the optic tectum (the number of the animals examined for each group is listed in the panel). Cytoplasmic and yolk injection results are presented for flt4 and ccbe1 dgRNPs. (D) Experimental workflow of the yolk microinjection experiments for panel (E). Injection cocktails containing Cas9 protein with and without the three vegfab crRNA:tracrRNA duplexes were injected into the yolk of one-cell stage Tg(kdrl:EGFP);Tg(kdrl:NLS-mCherry) embryos. Injected progeny were analyzed for VTA formation at 8 dpf. (E) Quantification of ECs at the VTAs within a 5 somite region per animal at 8 dpf after yolk injection of Cas9 protein with and without the three vegfab crRNA:tracrRNA duplexes (n = 20 for Cas9 controls and n = 33 for vegfab crRNA1-3). A two-tailed Student’s t-test was used to calculate P values. (F) Percentage of larvae of indicated treatment with and without VTAs within a 5 somite region per animal at 8 dpf (the number of the animals examined for each group is listed in the panel). Cytoplasmic and yolk injection results are presented for vegfab dgRNPs. (G) A summary table listing the percentage of larvae of indicated treatment that displayed biallelic mutant vascular phenotypes. A complete absence of FGPs at 6 dpf (flt4 and ccbe1), or of VTAs at 8 dpf (vegfab), were defined as biallelic mutant vascular phenotypes. (H) A summary table listing the percentage of unviable larvae following the indicated treatment. The number of the animals examined for each treatment is listed in the panel. Fisher’s exact test was used to determine significance between cytoplasmic and yolk injections for flt4, ccbe1, and vegfab in panels (C,F). However, no statistical difference was detected for any of the gene.
FIGURE 5 Yolk injections of three dgRNPs per gene allowed efficient generation of biallelic F0 knockouts but were less consistent than cytoplasmic injections. (A) Experimental workflow of the yolk microinjection experiments for panel (B). Injection cocktails containing Cas9 protein with and without the three flt4 or ccbe1 crRNA:tracrRNA duplexes were injected into the yolk of one-cell stage Tg(fli1:nEGFP);Tg(lyve1:DsRed) embryos. Injected progeny were analyzed at 6 dpf for FGP formation over the optic tectum. (B) Quantification of FGPs over the optic tectum at 6 dpf after yolk injection of Cas9 protein with and without the three flt4 or ccbe1 crRNA:tracrRNA duplexes (n = 20 for Cas9 controls, n = 35 for flt4 crRNA1-3 and ccbe1 crRNA1-3). All flt4 dgRNPs-injected larvae except one failed to form FGPs over the optic tectum. Approximately 89% of ccbe1 dgRNPs-injected larvae completely lacked FGPs, but the remaining fish formed FGPs in a manner comparable to Cas9-injected controls. A one-way ANOVA followed by Dunnett’s post hoc test was used to calculate P values. (C) Percentage of 6 dpf larvae of indicated treatment with and without FGPs over the optic tectum (the number of the animals examined for each group is listed in the panel). Cytoplasmic and yolk injection results are presented for flt4 and ccbe1 dgRNPs. (D) Experimental workflow of the yolk microinjection experiments for panel (E). Injection cocktails containing Cas9 protein with and without the three vegfab crRNA:tracrRNA duplexes were injected into the yolk of one-cell stage Tg(kdrl:EGFP);Tg(kdrl:NLS-mCherry) embryos. Injected progeny were analyzed for VTA formation at 8 dpf. (E) Quantification of ECs at the VTAs within a 5 somite region per animal at 8 dpf after yolk injection of Cas9 protein with and without the three vegfab crRNA:tracrRNA duplexes (n = 20 for Cas9 controls and n = 33 for vegfab crRNA1-3). A two-tailed Student’s t-test was used to calculate P values. (F) Percentage of larvae of indicated treatment with and without VTAs within a 5 somite region per animal at 8 dpf (the number of the animals examined for each group is listed in the panel). Cytoplasmic and yolk injection results are presented for vegfab dgRNPs. (G) A summary table listing the percentage of larvae of indicated treatment that displayed biallelic mutant vascular phenotypes. A complete absence of FGPs at 6 dpf (flt4 and ccbe1), or of VTAs at 8 dpf (vegfab), were defined as biallelic mutant vascular phenotypes. (H) A summary table listing the percentage of unviable larvae following the indicated treatment. The number of the animals examined for each treatment is listed in the panel. Fisher’s exact test was used to determine significance between cytoplasmic and yolk injections for flt4, ccbe1, and vegfab in panels (C,F). However, no statistical difference was detected for any of the gene.CC BY · PubMed Central
FIGURE 6 Analysis of large genomic deletions and mRNA decay following cytoplasmic dgRNP injections. (A) Diagram of part of the flt4 genomic locus, indicating approximate locations of the three crRNA target sites and PCR primers. Primer 1 (P1) and primer 2 (P2) were designed to amplify a 237 bp genomic fragment of WT alleles. Primer 3 (P3) was designed far from P1 so these primer pairs cannot efficiently amplify a large genomic fragment (∼30.8 kb) without undergoing genomic deletions (∼30.4 kb) induced by crRNA1 and crRNA3. (B) Diagram of the same part of the flt4 genomic locus after the anticipated genomic deletion (∼30.4 kb) induced by simultaneous targeting of crRNA1 and crRNA3. This genomic deletion is expected to enable PCR amplification of approximately 400 bp of the genomic fragment using primers P1 and P3, which flank these two crRNAs’ target sites. (C) Diagram of part of the vegfab genomic locus, indicating approximate locations of the three crRNA target sites and PCR primers. Primer 2 (P2) and primer 3 (P3) were designed to amplify a 268 bp genomic fragment of WT alleles. Primer 1 (P1) was designed far from P3 so these primer pairs cannot efficiently amplify a large genomic fragment (∼2.32 kb) without undergoing genomic deletions (∼1.89 kb) induced by crRNA1 and crRNA3. (D) Diagram of the same part of the vegfab genomic locus after the anticipated genomic deletion (∼1.89 kb) induced by simultaneous targeting of crRNA1 and crRNA3. This genomic deletion is expected to enable PCR amplification of approximately 430 bp of the genomic fragment using primers P1 and P3, which flank these two crRNAs’ target sites. (E, E’) Genomic DNA from uninjected larvae and those injected with the dgRNPs containing indicated flt4 crRNAs was amplified with the triple primer mix (P1 + P2 + P3; E). Uninjected larvae and those injected with individual crRNAs displayed only a 237 bp WT genomic fragment band. However, the WT band and an additional amplicon (asterisks) expected to be generated from genomic deletion alleles were both detected in the genomes of some F0 larvae injected with the triple dgRNPs. The additional amplicons were detected in more efficient manner with PCR using only the primer pairs (P1 + P3; E’). The bands marked by orange asterisks were detected at the approximate size of 400 bp, whereas those marked by green asterisks were observed at around 500 bp. These bands were cut out and sequenced. (F,F’) Genomic DNA from uninjected larvae and those injected with the dgRNPs containing indicated vegfab crRNAs was amplified with the triple primer mix (P1 + P2 + P3; F). Uninjected larvae and those injected with individual crRNAs displayed only a 268 bp WT genomic fragment band. However, the WT band and an additional amplicon (asterisks) expected to be generated from genomic deletion alleles were both detected in the genomes of all F0 larvae injected with the triple dgRNPs. The additional amplicons were detected at the approximate size of 430 bp with PCR using only the primer pairs (P1 + P3; F’). These bands were cut out and sequenced. (G) Sequence analysis of the PCR products indicated by orange asterisks in (E’) from the triple flt4 dgRNP-injected larvae. The sequence indicated by ΔcrRNA1/crRNA3 represents expected perfect end joining after simultaneous flt4 crRNA1 and crRNA3 gene targeting. The number of base pairs that were apparently deleted from this perfect end joining of predicted cleavage sites is indicated. crRNA1 and crRNA3 target sites are indicated in blue and pink, respectively, along with their PAM sequences (green). (H) Sequence analysis of the PCR products indicated by orange asterisks in (F’) from the triple vegfab dgRNP-injected larvae. The sequence results from the larvae #1–3 are presented. The sequence indicated by ΔcrRNA1/crRNA3 represents expected perfect end joining after simultaneous vegfab crRNA1 and crRNA3 gene targeting. The number of base pairs that were apparently deleted from this perfect end joining of predicted cleavage sites is indicated. crRNA1 and crRNA3 target sites are indicated in blue and pink, respectively, along with their PAM sequences (green). (I) qPCR analysis of flt4 mRNA expression levels in 26 hpf embryonic samples of the indicated treatments. Triple dgRNP-injected samples showed significantly reduced flt4 mRNA levels compared to uninjected and Cas9 injected controls. n = 3 biologically independent samples. (J) qPCR analysis of vegfab mRNA expression levels in 26 hpf embryonic samples of the indicated treatments. No significant difference in vegfab mRNA levels was observed across the groups. n = 3 biologically independent samples. For qPCR analyses (I,J), uninjected expression levels were set at 1. A one-way ANOVA followed by Tukey’s post hoc test was used to calculate P values.
FIGURE 6 Analysis of large genomic deletions and mRNA decay following cytoplasmic dgRNP injections. (A) Diagram of part of the flt4 genomic locus, indicating approximate locations of the three crRNA target sites and PCR primers. Primer 1 (P1) and primer 2 (P2) were designed to amplify a 237 bp genomic fragment of WT alleles. Primer 3 (P3) was designed far from P1 so these primer pairs cannot efficiently amplify a large genomic fragment (∼30.8 kb) without undergoing genomic deletions (∼30.4 kb) induced by crRNA1 and crRNA3. (B) Diagram of the same part of the flt4 genomic locus after the anticipated genomic deletion (∼30.4 kb) induced by simultaneous targeting of crRNA1 and crRNA3. This genomic deletion is expected to enable PCR amplification of approximately 400 bp of the genomic fragment using primers P1 and P3, which flank these two crRNAs’ target sites. (C) Diagram of part of the vegfab genomic locus, indicating approximate locations of the three crRNA target sites and PCR primers. Primer 2 (P2) and primer 3 (P3) were designed to amplify a 268 bp genomic fragment of WT alleles. Primer 1 (P1) was designed far from P3 so these primer pairs cannot efficiently amplify a large genomic fragment (∼2.32 kb) without undergoing genomic deletions (∼1.89 kb) induced by crRNA1 and crRNA3. (D) Diagram of the same part of the vegfab genomic locus after the anticipated genomic deletion (∼1.89 kb) induced by simultaneous targeting of crRNA1 and crRNA3. This genomic deletion is expected to enable PCR amplification of approximately 430 bp of the genomic fragment using primers P1 and P3, which flank these two crRNAs’ target sites. (E, E’) Genomic DNA from uninjected larvae and those injected with the dgRNPs containing indicated flt4 crRNAs was amplified with the triple primer mix (P1 + P2 + P3; E). Uninjected larvae and those injected with individual crRNAs displayed only a 237 bp WT genomic fragment band. However, the WT band and an additional amplicon (asterisks) expected to be generated from genomic deletion alleles were both detected in the genomes of some F0 larvae injected with the triple dgRNPs. The additional amplicons were detected in more efficient manner with PCR using only the primer pairs (P1 + P3; E’). The bands marked by orange asterisks were detected at the approximate size of 400 bp, whereas those marked by green asterisks were observed at around 500 bp. These bands were cut out and sequenced. (F,F’) Genomic DNA from uninjected larvae and those injected with the dgRNPs containing indicated vegfab crRNAs was amplified with the triple primer mix (P1 + P2 + P3; F). Uninjected larvae and those injected with individual crRNAs displayed only a 268 bp WT genomic fragment band. However, the WT band and an additional amplicon (asterisks) expected to be generated from genomic deletion alleles were both detected in the genomes of all F0 larvae injected with the triple dgRNPs. The additional amplicons were detected at the approximate size of 430 bp with PCR using only the primer pairs (P1 + P3; F’). These bands were cut out and sequenced. (G) Sequence analysis of the PCR products indicated by orange asterisks in (E’) from the triple flt4 dgRNP-injected larvae. The sequence indicated by ΔcrRNA1/crRNA3 represents expected perfect end joining after simultaneous flt4 crRNA1 and crRNA3 gene targeting. The number of base pairs that were apparently deleted from this perfect end joining of predicted cleavage sites is indicated. crRNA1 and crRNA3 target sites are indicated in blue and pink, respectively, along with their PAM sequences (green). (H) Sequence analysis of the PCR products indicated by orange asterisks in (F’) from the triple vegfab dgRNP-injected larvae. The sequence results from the larvae #1–3 are presented. The sequence indicated by ΔcrRNA1/crRNA3 represents expected perfect end joining after simultaneous vegfab crRNA1 and crRNA3 gene targeting. The number of base pairs that were apparently deleted from this perfect end joining of predicted cleavage sites is indicated. crRNA1 and crRNA3 target sites are indicated in blue and pink, respectively, along with their PAM sequences (green). (I) qPCR analysis of flt4 mRNA expression levels in 26 hpf embryonic samples of the indicated treatments. Triple dgRNP-injected samples showed significantly reduced flt4 mRNA levels compared to uninjected and Cas9 injected controls. n = 3 biologically independent samples. (J) qPCR analysis of vegfab mRNA expression levels in 26 hpf embryonic samples of the indicated treatments. No significant difference in vegfab mRNA levels was observed across the groups. n = 3 biologically independent samples. For qPCR analyses (I,J), uninjected expression levels were set at 1. A one-way ANOVA followed by Tukey’s post hoc test was used to calculate P values.CC BY · PubMed Central
FIGURE 7 Cytoplasmic injections of two dgRNPs per gene led to variable efficiency of biallelic gene disruptions in F0 animals. (A) Experimental workflow of the cytoplasmic microinjection experiments for panel (B). Injection cocktails containing Cas9 protein with two unique flt4 crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage Tg(fli1:nEGFP);Tg(lyve1:DsRed) embryos. Injected progeny were analyzed at 6 dpf for FGP formation over the optic tectum. (B) Quantification of FGPs over the optic tectum at 6 dpf after cytoplasmic injection of Cas9 protein with the two indicated flt4 crRNA:tracrRNA duplexes (n = 20 for uninjected controls; and n = 32 for flt4 crRNAs1&2, crRNAs1&3, and crRNAs2&3). Injections of two unique flt4 dgRNPs led to varying degrees of FGP loss over the optic tectum. (C) Experimental workflow of the cytoplasmic microinjection experiments for panel (D). Injection cocktails containing Cas9 protein with two unique vegfab crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage Tg(kdrl:EGFP);Tg(kdrl:NLS-mCherry) embryos. Injected progeny were analyzed for VTA formation at 8 dpf. (D) Quantification of ECs at the VTAs within a 5 somite region per animal at 8 dpf after yolk injection of Cas9 protein with the two indicated vegfab crRNA:tracrRNA duplexes (n = 20 for uninjected controls; and n = 32 for vegfab crRNAs1&2, crRNAs1&3, and crRNAs2&3). (E) A summary table listing the percentage of larvae of the indicated treatment that displayed biallelic mutant vascular phenotypes. A complete absence of FGPs at 6 dpf (flt4), or of VTAs at 8 dpf (vegfab), were defined as biallelic mutant vascular phenotypes. (F) A summary table listing the percentage of unviable larvae following the indicated treatment. The number of the animals examined for each treatment is listed in the panel. A one-way ANOVA followed by Tukey’s post hoc test was used to calculate P values for panels (B,D).
FIGURE 7 Cytoplasmic injections of two dgRNPs per gene led to variable efficiency of biallelic gene disruptions in F0 animals. (A) Experimental workflow of the cytoplasmic microinjection experiments for panel (B). Injection cocktails containing Cas9 protein with two unique flt4 crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage Tg(fli1:nEGFP);Tg(lyve1:DsRed) embryos. Injected progeny were analyzed at 6 dpf for FGP formation over the optic tectum. (B) Quantification of FGPs over the optic tectum at 6 dpf after cytoplasmic injection of Cas9 protein with the two indicated flt4 crRNA:tracrRNA duplexes (n = 20 for uninjected controls; and n = 32 for flt4 crRNAs1&2, crRNAs1&3, and crRNAs2&3). Injections of two unique flt4 dgRNPs led to varying degrees of FGP loss over the optic tectum. (C) Experimental workflow of the cytoplasmic microinjection experiments for panel (D). Injection cocktails containing Cas9 protein with two unique vegfab crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage Tg(kdrl:EGFP);Tg(kdrl:NLS-mCherry) embryos. Injected progeny were analyzed for VTA formation at 8 dpf. (D) Quantification of ECs at the VTAs within a 5 somite region per animal at 8 dpf after yolk injection of Cas9 protein with the two indicated vegfab crRNA:tracrRNA duplexes (n = 20 for uninjected controls; and n = 32 for vegfab crRNAs1&2, crRNAs1&3, and crRNAs2&3). (E) A summary table listing the percentage of larvae of the indicated treatment that displayed biallelic mutant vascular phenotypes. A complete absence of FGPs at 6 dpf (flt4), or of VTAs at 8 dpf (vegfab), were defined as biallelic mutant vascular phenotypes. (F) A summary table listing the percentage of unviable larvae following the indicated treatment. The number of the animals examined for each treatment is listed in the panel. A one-way ANOVA followed by Tukey’s post hoc test was used to calculate P values for panels (B,D).CC BY · PubMed Central
FIGURE 8 Simultaneous disruptions of the Vegfr2 zebrafish paralog genes using a triple dgRNP injection method. (A) Experimental workflow of the phenotypic analysis presented in panels (B–I). Adult kdr+/–;kdrl+/– fish carrying the Tg(kdrl:EGFP) transgene were incrossed, and progeny generated from the crosses were imaged to quantify the formation of the dorsal aorta and aISVs at 32 hpf. (B–G) Lateral views of kdr+/+ (B), kdr+/– (C), kdr–/– (D), kdr+/+;kdrl–/– (E), kdr+/–;kdrl–/– (F), and kdr–/–;kdrl–/– (G) trunk vasculature visualized by Tg(kdrl:EGFP) expression at 32 hpf. No obvious trunk vascular defects were observed in kdr+/+, kdr+/–, and kdr–/– embryos (B–D). kdr+/+;kdrl–/– embryos exhibited stalled aISV growth (E), and this aISV growth defect was exacerbated in kdr+/–;kdrl–/– embryos (F). In all kdr–/–;kdrl–/– embryos examined at 32 hpf, the dorsal aorta failed to form (G). DA: dorsal aorta; PCV: posterior cardinal vein. (H) Percentage of 32 hpf embryos of indicated genotype exhibiting the presence, partial presence, and absence of the dorsal aorta (the number of the animals examined per genotype is listed in the panel). (I) Quantification of aISV lengths of 32 hpf embryos of the indicated genotypes (n = 10 for kdr+/+, n = 23 for kdr+/–, n = 16 for kdr–/–, n = 17 for kdr+/+;kdrl–/–, and n = 30 for kdr+/–;kdrl–/–; 5 aISV lengths measured per embryo). kdr+/–;kdrl–/– embryos displayed significantly shorter average aISV lengths than those of kdr+/+;kdrl–/– embryos. (J) Experimental workflow of the microinjection experiments for panels (K–R). Injection cocktails containing Cas9 protein with and without the three kdrl crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage embryos generated from incrosses of kdr+/– fish carrying the Tg(kdrl:EGFP) transgene. Injected progeny were imaged to quantify the formation of the dorsal aorta and aISVs at 32 hpf. (K–P) Lateral trunk views of the 32 hpf kdr+/+ (K,N), kdr+/– (L,O), and kdr–/– (M,P) embryos injected with Cas9 protein with (N–P) or without (K–M) the three kdrl crRNA:tracrRNA duplexes (kdrl dgRNPs). Trunk vasculature was visualized by Tg(kdrl:EGFP) expression. No obvious trunk vascular defects were observed in kdr+/+, kdr+/–, and kdr–/– embryos injected with Cas9 (K–M). In contrast, kdr+/+ embryos injected with the triple kdrl dgRNPs displayed the stalled aISV phenotype (N). Exacerbated aISV defects were observed in the kdrl dgRNPs-injected kdr+/– embryos (O). A nearly half of the kdrl dgRNPs-injected kdr–/– embryos exhibited a complete absence of the dorsal aorta (P). (Q) Percentage of 32 hpf embryos of the indicated genotype and treatment exhibiting the presence, partial presence, and absence of the dorsal aorta (the number of the animals examined per group is listed in the panel). (R) Quantification of aISV lengths of 32 hpf embryos of the indicated genotype and treatment (n = 11 for Cas9-injected kdr+/+, n = 27 for Cas9-injected kdr+/–, n = 17 for Cas9-injected kdr–/–, n = 31 for kdrl dgRNPs-injected kdr+/+, and n = 47 for kdrl dgRNPs-injected kdr+/–; 5 aISV lengths per embryo measured). kdr+/–embryos injected with the kdrl dgRNPs exhibited significantly shorter average aISV lengths than those of the same dgRNPs-injected kdr+/+ fish. (S) Experimental workflow of the microinjection experiments for panels (T–W). Injection cocktails containing Cas9 protein with and without the three kdr/kdrl crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage embryos generated from incrosses of fish carrying the Tg(kdrl:EGFP) transgene. Injected progeny were imaged to observe the formation of the dorsal aorta and aISVs at 32 hpf. (T–Y) Lateral trunk views of the 32 hpf Tg(kdrl:EGFP) embryos injected with Cas9 protein (T) or with the three kdr (U) or kdrl (X) dgRNPs, or the combined six kdr and kdrl dgRNPs (Y). No obvious trunk vascular defects were observed in embryos injected with Cas9 (T) and the three kdr dgRNPs (U). In contrast, kdrl dgRNPs-injected embryos displayed the stalled aISV phenotype (X). The combined injections of the kdr and kdrl dgRNPs led to a complete absence of the dorsal aorta (Y) as observed in 32 hpf kdr–/–;kdrl–/– embryos. (Z) Percentage of 32 hpf embryos of the indicated treatment exhibiting the presence, partial presence, and absence of the dorsal aorta (the number of the animals examined per group is listed in the panel). (V) Percentage of 32 hpf embryos exhibiting an absence of the dorsal aorta following the cytoplasmic injections of kdr or kdrl triple dgRNPs, or these combined six dgRNPs (the number of the animals examined per group is listed in the panel). (W) Percentage of 24 hpf dead embryos following the cytoplasmic injections of kdr or kdrl triple dgRNPs, or these combined six dgRNPs (the number of the animals examined per group is listed in the panel). A one-way ANOVA followed by Tukey’s post hoc test was used to calculate P values for panels (I,R). Fisher’s exact test was used to calculate P values for panels (H,Q,Z). Scale bars: 50 μm.
FIGURE 8 Simultaneous disruptions of the Vegfr2 zebrafish paralog genes using a triple dgRNP injection method. (A) Experimental workflow of the phenotypic analysis presented in panels (B–I). Adult kdr+/–;kdrl+/– fish carrying the Tg(kdrl:EGFP) transgene were incrossed, and progeny generated from the crosses were imaged to quantify the formation of the dorsal aorta and aISVs at 32 hpf. (B–G) Lateral views of kdr+/+ (B), kdr+/– (C), kdr–/– (D), kdr+/+;kdrl–/– (E), kdr+/–;kdrl–/– (F), and kdr–/–;kdrl–/– (G) trunk vasculature visualized by Tg(kdrl:EGFP) expression at 32 hpf. No obvious trunk vascular defects were observed in kdr+/+, kdr+/–, and kdr–/– embryos (B–D). kdr+/+;kdrl–/– embryos exhibited stalled aISV growth (E), and this aISV growth defect was exacerbated in kdr+/–;kdrl–/– embryos (F). In all kdr–/–;kdrl–/– embryos examined at 32 hpf, the dorsal aorta failed to form (G). DA: dorsal aorta; PCV: posterior cardinal vein. (H) Percentage of 32 hpf embryos of indicated genotype exhibiting the presence, partial presence, and absence of the dorsal aorta (the number of the animals examined per genotype is listed in the panel). (I) Quantification of aISV lengths of 32 hpf embryos of the indicated genotypes (n = 10 for kdr+/+, n = 23 for kdr+/–, n = 16 for kdr–/–, n = 17 for kdr+/+;kdrl–/–, and n = 30 for kdr+/–;kdrl–/–; 5 aISV lengths measured per embryo). kdr+/–;kdrl–/– embryos displayed significantly shorter average aISV lengths than those of kdr+/+;kdrl–/– embryos. (J) Experimental workflow of the microinjection experiments for panels (K–R). Injection cocktails containing Cas9 protein with and without the three kdrl crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage embryos generated from incrosses of kdr+/– fish carrying the Tg(kdrl:EGFP) transgene. Injected progeny were imaged to quantify the formation of the dorsal aorta and aISVs at 32 hpf. (K–P) Lateral trunk views of the 32 hpf kdr+/+ (K,N), kdr+/– (L,O), and kdr–/– (M,P) embryos injected with Cas9 protein with (N–P) or without (K–M) the three kdrl crRNA:tracrRNA duplexes (kdrl dgRNPs). Trunk vasculature was visualized by Tg(kdrl:EGFP) expression. No obvious trunk vascular defects were observed in kdr+/+, kdr+/–, and kdr–/– embryos injected with Cas9 (K–M). In contrast, kdr+/+ embryos injected with the triple kdrl dgRNPs displayed the stalled aISV phenotype (N). Exacerbated aISV defects were observed in the kdrl dgRNPs-injected kdr+/– embryos (O). A nearly half of the kdrl dgRNPs-injected kdr–/– embryos exhibited a complete absence of the dorsal aorta (P). (Q) Percentage of 32 hpf embryos of the indicated genotype and treatment exhibiting the presence, partial presence, and absence of the dorsal aorta (the number of the animals examined per group is listed in the panel). (R) Quantification of aISV lengths of 32 hpf embryos of the indicated genotype and treatment (n = 11 for Cas9-injected kdr+/+, n = 27 for Cas9-injected kdr+/–, n = 17 for Cas9-injected kdr–/–, n = 31 for kdrl dgRNPs-injected kdr+/+, and n = 47 for kdrl dgRNPs-injected kdr+/–; 5 aISV lengths per embryo measured). kdr+/–embryos injected with the kdrl dgRNPs exhibited significantly shorter average aISV lengths than those of the same dgRNPs-injected kdr+/+ fish. (S) Experimental workflow of the microinjection experiments for panels (T–W). Injection cocktails containing Cas9 protein with and without the three kdr/kdrl crRNA:tracrRNA duplexes were injected into the cytoplasm of one-cell stage embryos generated from incrosses of fish carrying the Tg(kdrl:EGFP) transgene. Injected progeny were imaged to observe the formation of the dorsal aorta and aISVs at 32 hpf. (T–Y) Lateral trunk views of the 32 hpf Tg(kdrl:EGFP) embryos injected with Cas9 protein (T) or with the three kdr (U) or kdrl (X) dgRNPs, or the combined six kdr and kdrl dgRNPs (Y). No obvious trunk vascular defects were observed in embryos injected with Cas9 (T) and the three kdr dgRNPs (U). In contrast, kdrl dgRNPs-injected embryos displayed the stalled aISV phenotype (X). The combined injections of the kdr and kdrl dgRNPs led to a complete absence of the dorsal aorta (Y) as observed in 32 hpf kdr–/–;kdrl–/– embryos. (Z) Percentage of 32 hpf embryos of the indicated treatment exhibiting the presence, partial presence, and absence of the dorsal aorta (the number of the animals examined per group is listed in the panel). (V) Percentage of 32 hpf embryos exhibiting an absence of the dorsal aorta following the cytoplasmic injections of kdr or kdrl triple dgRNPs, or these combined six dgRNPs (the number of the animals examined per group is listed in the panel). (W) Percentage of 24 hpf dead embryos following the cytoplasmic injections of kdr or kdrl triple dgRNPs, or these combined six dgRNPs (the number of the animals examined per group is listed in the panel). A one-way ANOVA followed by Tukey’s post hoc test was used to calculate P values for panels (I,R). Fisher’s exact test was used to calculate P values for panels (H,Q,Z). Scale bars: 50 μm.CC BY · PubMed Central
FIGURE 9 Flowchart summary of triple dgRNP injections for rapid F0 phenotypic screens. This flowchart serves as a quick snapshot of experimental workflow and estimated timelines of the presented method. This method is designed to minimize users’ efforts for crRNA selection, validation, and efficacy testing while maximizing the chance of biallelic disruptions of target genes. Refer to the materials and methods section for detailed methodological descriptions of each step in this flowchart.
FIGURE 9 Flowchart summary of triple dgRNP injections for rapid F0 phenotypic screens. This flowchart serves as a quick snapshot of experimental workflow and estimated timelines of the presented method. This method is designed to minimize users’ efforts for crRNA selection, validation, and efficacy testing while maximizing the chance of biallelic disruptions of target genes. Refer to the materials and methods section for detailed methodological descriptions of each step in this flowchart.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 65 references

Metadata

Authors
Quick, R. E., Buck, L. D., Parab, S., Tolbert, Z. R., & Matsuoka, R. L.
Affiliations
Cleveland Clinic (US)
Corresponding author
Matsuoka, Ryota L.
ORCID
Venue
Frontiers in Cell and Developmental Biology
Publisher
Frontiers Media SA
Published
2021-10-22
Volume
9
Pages
735598-735598
ISSN
2296-634X (ISSN-L)
DOI
10.3389/fcell.2021.735598
PMC
PMC8570140
OpenAlex
W3208177987
Cited by
32
References
65
Citations by year
2026: 8 · 2025: 10 · 2024: 3 · 2023: 6 · 2022: 5
FWCI
1.10
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Cell and Developmental Biology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Institutes of Health
  • Cleveland Clinic
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Zebrafish Biomedical Research Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
Keywords
CRISPR/Cas9 mutagenesis, ribonucleoprotein, cardiovascular phenotyping, gene redundancy, frameshift mutation, mRNA degradation, cytoplasmic injection, yolk injection

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review

How to cite

Cite CoChat for the checks on this page, and the paper for its own findings.

This page
CoChat. (2026). Citation check: Highly Efficient Synthetic CRISPR RNA/Cas9-Based Mutagenesis for Rapid Cardiovascular Phenotypic Screening in F0 Zebrafish. https://app.cochat.ai/papers/10.3389/fcell.2021.735598
The paper
Quick, R. E., Buck, L. D., Parab, S., Tolbert, Z. R., & Matsuoka, R. L. (2021). Highly Efficient Synthetic CRISPR RNA/Cas9-Based Mutagenesis for Rapid Cardiovascular Phenotypic Screening in F0 Zebrafish. Frontiers in Cell and Developmental Biology. https://doi.org/10.3389/fcell.2021.735598

Data sources

Bibliographic data: Semantic Scholar, OpenAlex, Crossref.

Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).

Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.