Journal: bioRxiv

Article Title: Highly efficient synthetic CRISPR RNA/Cas9-based mutagenesis for rapid cardiovascular phenotypic screening in F0 zebrafish

doi: 10.1101/2021.07.01.450753

Figure Lengend Snippet: ( 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 and without 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 and 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 , and T ; 500 µm in P .

Article Snippet: Cas9 nuclease, V3, IDT) was prepared by diluting a 62 μM original Cas9 protein sample from IDT with Cas9 buffer (20 mM HEPES-NaOH (pH 7.5), 350 mM KCl, and 20% glycerol).

Techniques: Generated, Expressing, CRISPR, Injection, Genomic Sequencing

Journal: bioRxiv

Article Title: Highly efficient synthetic CRISPR RNA/Cas9-based mutagenesis for rapid cardiovascular phenotypic screening in F0 zebrafish

doi: 10.1101/2021.07.01.450753

Figure Lengend Snippet: ( 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 and 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 and 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 and P ), those injected with the three flt4 dgRNPs displayed a complete lack of the FGPs at both stages ( O and 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 , and Q ; 1 mm in M .

Article Snippet: Cas9 nuclease, V3, IDT) was prepared by diluting a 62 μM original Cas9 protein sample from IDT with Cas9 buffer (20 mM HEPES-NaOH (pH 7.5), 350 mM KCl, and 20% glycerol).

Techniques: Injection, Genomic Sequencing

Journal: bioRxiv

Article Title: Highly efficient synthetic CRISPR RNA/Cas9-based mutagenesis for rapid cardiovascular phenotypic screening in F0 zebrafish

doi: 10.1101/2021.07.01.450753

Figure Lengend Snippet: ( 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 and 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 and 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 and P ). However, fish injected with the three ccbe1 dgRNPs completely lacked the FGPs at both stages ( O and 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 , and Q ; 1 mm in M .

Article Snippet: Cas9 nuclease, V3, IDT) was prepared by diluting a 62 μM original Cas9 protein sample from IDT with Cas9 buffer (20 mM HEPES-NaOH (pH 7.5), 350 mM KCl, and 20% glycerol).

Techniques: Binding Assay, Injection, Genomic Sequencing

Journal: bioRxiv

Article Title: Highly efficient synthetic CRISPR RNA/Cas9-based mutagenesis for rapid cardiovascular phenotypic screening in F0 zebrafish

doi: 10.1101/2021.07.01.450753

Figure Lengend Snippet: ( 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 Vegfab 171 isoform. Vegfab 171 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 and 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 and P ), those injected with the three vegfab dgRNPs completely lacked VTAs at both stages ( O and 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 , and Q ; 1 mm in M .

Article Snippet: Cas9 nuclease, V3, IDT) was prepared by diluting a 62 μM original Cas9 protein sample from IDT with Cas9 buffer (20 mM HEPES-NaOH (pH 7.5), 350 mM KCl, and 20% glycerol).

Techniques: Binding Assay, Injection, Genomic Sequencing

Journal: bioRxiv

Article Title: Highly efficient synthetic CRISPR RNA/Cas9-based mutagenesis for rapid cardiovascular phenotypic screening in F0 zebrafish

doi: 10.1101/2021.07.01.450753

Figure Lengend Snippet: ( 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.

Article Snippet: Cas9 nuclease, V3, IDT) was prepared by diluting a 62 μM original Cas9 protein sample from IDT with Cas9 buffer (20 mM HEPES-NaOH (pH 7.5), 350 mM KCl, and 20% glycerol).

Techniques: Injection, Two Tailed Test, Mutagenesis

Journal: bioRxiv

Article Title: Highly efficient synthetic CRISPR RNA/Cas9-based mutagenesis for rapid cardiovascular phenotypic screening in F0 zebrafish

doi: 10.1101/2021.07.01.450753

Figure Lengend Snippet: ( 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 and 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 and 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 genetic 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 genetic targeting. The number of base pairs that were apparently deleted from to 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 and 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.

Article Snippet: Cas9 nuclease, V3, IDT) was prepared by diluting a 62 μM original Cas9 protein sample from IDT with Cas9 buffer (20 mM HEPES-NaOH (pH 7.5), 350 mM KCl, and 20% glycerol).

Techniques: Amplification, Injection, Generated, Sequencing, Expressing

Journal: bioRxiv

Article Title: Highly efficient synthetic CRISPR RNA/Cas9-based mutagenesis for rapid cardiovascular phenotypic screening in F0 zebrafish

doi: 10.1101/2021.07.01.450753

Figure Lengend Snippet: ( A ) Experimental workflow of the cytoplasmic microinjection experiments for panel ( B ). Injection cocktails containing Cas9 protein with and without 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 and without 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 and without 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 and without 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 ).

Article Snippet: Cas9 nuclease, V3, IDT) was prepared by diluting a 62 μM original Cas9 protein sample from IDT with Cas9 buffer (20 mM HEPES-NaOH (pH 7.5), 350 mM KCl, and 20% glycerol).

Techniques: Injection, Mutagenesis

Journal: bioRxiv

Article Title: Highly efficient synthetic CRISPR RNA/Cas9-based mutagenesis for rapid cardiovascular phenotypic screening in F0 zebrafish

doi: 10.1101/2021.07.01.450753

Figure Lengend Snippet: ( 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 with ( U – Y ) or without ( T ) the three kdr or kdrl crRNA:tracrRNA duplexes (crRNAs), or the combined six kdr and kdrl crRNAs. 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.

Article Snippet: Cas9 nuclease, V3, IDT) was prepared by diluting a 62 μM original Cas9 protein sample from IDT with Cas9 buffer (20 mM HEPES-NaOH (pH 7.5), 350 mM KCl, and 20% glycerol).

Techniques: Generated, Expressing, Injection

Journal: bioRxiv

Article Title: Highly efficient synthetic CRISPR RNA/Cas9-based mutagenesis for rapid cardiovascular phenotypic screening in F0 zebrafish

doi: 10.1101/2021.07.01.450753

Figure Lengend Snippet: ( A ) Three synthetic crRNAs were designed to target sequences within exon 1 (E1), E3, and E5 on the kdr genomic locus. ( B ) Predicted domain structure of zebrafish Kdr. Kdr 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. ( C – E ) HRMA used to validate the efficacy of the three designed crRNAs. The melting curves of 6 independent embryos injected with the dgRNP complex containing kdr crRNA1 ( C , pink ), crRNA2 ( D , orange ), or crRNA3 ( E , purple ) are presented. The melting curves of 2 independent, uninjected ( C – E , blue ) and Cas9-injected ( C – E , green ) sibling embryos are also presented for comparison for each crRNA. Injection of each dgRNP cocktail disrupted the corresponding target genomic sequences.

Article Snippet: Cas9 nuclease, V3, IDT) was prepared by diluting a 62 μM original Cas9 protein sample from IDT with Cas9 buffer (20 mM HEPES-NaOH (pH 7.5), 350 mM KCl, and 20% glycerol).

Techniques: Injection, Genomic Sequencing

Journal: bioRxiv

Article Title: Towards CRISPR/Cas9-based gene drive in the diamondback moth Plutella xylostella

doi: 10.1101/2021.10.05.462963

Figure Lengend Snippet: A: Donor cassettes for piggyBac -mediated transformation of Cas9 lines. B: HDR-based integration of homing elements into endogenous marker genes Pxyellow and Pxkmo .

Article Snippet: The Cas9 Sequencing Kit (Oxford Nanopore Technology) was used to prepare a library targeting the insertion.

Techniques: Transformation Assay, Marker

Journal: bioRxiv

Article Title: Towards CRISPR/Cas9-based gene drive in the diamondback moth Plutella xylostella

doi: 10.1101/2021.10.05.462963

Figure Lengend Snippet: A: Parental Cas9 lines were crossed with the 1619P15 line integrated into and expressing sgRNAs targeting Pxyellow . B: Parental Cas9 lines are crossed with the 1963C line integrated into and expressing sgRNAs targeting Pxkmo . Mosaic phenotypes are indicated using white arrows.

Article Snippet: The Cas9 Sequencing Kit (Oxford Nanopore Technology) was used to prepare a library targeting the insertion.

Techniques: Expressing

Journal: bioRxiv

Article Title: Towards CRISPR/Cas9-based gene drive in the diamondback moth Plutella xylostella

doi: 10.1101/2021.10.05.462963

Figure Lengend Snippet: A: Proportion of F 2 individuals inheriting the relevant homing element. Offspring come from either a male (blue) or female (orange) Cas9-bearing F 1 parent. Due to non-significant effect of ‘grandparental’ (F 0 ) sex on the model, this factor has been collapsed in the data shown. Central tendency and error bars represent estimated mean proportions and associated approximate 95% confidence intervals for that treatment. Shaded areas (violin plots) represent the density distribution of the raw data. B: Germline cleavage efficiency (%) = no. phenotypic mutants (e.g. yellow eyes) / (no. phenotypic mutants + phenotypic wildtypes (e.g. black eyes)). Offspring come from either a male (blue) or female (orange) Cas9-bearing F 1 parent Data are plotted as means overlaid on raw data. For comparison of lines within the same sex, those which share a letter above the plotted data (e.g. ab and bc) are not significantly different from each other. For comparisons between sex, within each line, “*” shows the significant difference between subgroups. *: P < 0.05. **: P < 0.01. ***: P < 0.001.

Article Snippet: The Cas9 Sequencing Kit (Oxford Nanopore Technology) was used to prepare a library targeting the insertion.

Techniques: Comparison

Journal: Cell Metabolism

Article Title: A Role for p53 in the Adaptation to Glutamine Starvation through the Expression of SLC1A3

doi: 10.1016/j.cmet.2018.07.005

Figure Lengend Snippet: SLC1A3 Is a p53 Target that Sustains the Growth of Cancer Cell Lines under Glutamine Deprivation (A) SLC1A3 transcriptional expression in HCT116 WT and p53-null clones grown for 2 days in glutamine-free medium. Data are presented as mean ± SEM (averages of triplicate wells). (B) Western blots show SLC1A3 expression in HCT116 WT and p53-null clones grown for 2 days in glutamine-free medium. Note that AGC2 and SLC1A3 ( Figure S7 D) were probed on the same membrane, so the same ACTIN western blot is shown here and in Figure S7 D. (C and D) HCT116 WT cells were transiently depleted of SLC1A3 using siRNA, and proliferation assessed in complete medium (C) or in glutamine-free medium (D). Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). The downregulation of SLC1A3 was confirmed by western blot (D, right panel). Note that siCTR and siSLC1A3 samples were run on the same gel but intervening lanes have been removed (cut indicated by gray line). (E) MDA-MB-468, OVCAR-4, and IGROV1 cells infected with Cas9/SLC1A3 single guide RNA were cultured in glutamine-free medium for 2 days. Western blot shows efficient SLC1A3 depletion in Cas9/SLC1A3-infected cells. (F) Proliferation rates of cells cultured in complete medium (top panel) or in glutamine-free medium (bottom panel). Data are presented as mean ± SEM of one representative experiment (average of triplicate wells). (G) BALB/c nude mice were subcutaneously injected with MDA-MB-468 NTC cells (n = 6) or SLC1A3 KO cells (n = 8). Once the tumors were palpable, tumor volumes were measured twice a week by caliper measurement. Data are presented as mean ± SEM ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, two-way ANOVA plus Bonferroni’s post hoc test). (H) CD-1 nude mice were subcutaneously injected with IGROV1 NTC cells (n = 9) or SLC1A3 KO cells (n = 10). Once the tumors were palpable, tumor volumes were measured twice a week by caliper measurement. Data are presented as mean ± SEM ( ∗ p < 0.05, two-way ANOVA plus Bonferroni’s post hoc test). See also and .

Article Snippet: Human SLC1A3 was cloned into the pBABE-hygro vector (gift from Feng Zhang (Addgene plasmid # 1765; ): pBABE-hygro vector was digested with BamHI and SalI and cDNA coding for human SLC1A3 (purchased from IDT) cloned using In-Fusion HD cloning kit from Clontech (Takara Bio USA, Inc).

Techniques: Expressing, Clone Assay, Western Blot, Infection, Cell Culture, Injection

Journal: Cell Metabolism

Article Title: A Role for p53 in the Adaptation to Glutamine Starvation through the Expression of SLC1A3

doi: 10.1016/j.cmet.2018.07.005

Figure Lengend Snippet: SLC1A3 Depletion Reduces Aspartate Uptake and TCA Activity under Glutamine Deprivation, Phenocopying Loss of p53 (A) HCT116 WT cells transiently depleted of SLC1A3 using siRNA were grown for 2 days in glutamine-free medium and pulsed with [U- 13 C]aspartate for the final 24 hr. Extracellular levels of aspartate (m+4), normalized to cell number, were quantified over 24 hr. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (B) IGROV1, MDA-MB-468, and OVCAR4 WT and SLC1A3 KO cells were grown for 3 days in glutamine-free medium and pulsed with [U- 13 C]aspartate for the final 16 hr. Extracellular levels of aspartate (m+4), normalized to cell number, were quantified over 16 hr. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (C) IGROV1, MDA-MB-468, and OVCAR4 WT and SLC1A3 KO cells were grown for 3 days in glutamine-free medium and pulsed with [U- 13 C]aspartate for the final 16 hr. Intracellular aspartate level was analyzed. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (D–F) HCT116 WT cells transiently depleted of SLC1A3 using siRNA were grown for 2 days in glutamine-free medium and pulsed with [U- 13 C]aspartate for the final 16 hr. Stable isotopomer tracing analysis of intracellular aspartate (D), TCA-cycle intermediates (E), and glutamate and glutamine levels (F) is shown. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (G and H) IGROV1, MDA-MB-468, and OVCAR4 WT and SLC1A3 KO cells were grown for 3 days in glutamine-free medium and pulsed with [U- 13 C]aspartate for the final 16 hr. Stable isotopomer tracing analysis of intracellular TCA-cycle intermediates (G) and glutamate and glutamine levels (H) is shown. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). See also Figure S6 .

Article Snippet: Human SLC1A3 was cloned into the pBABE-hygro vector (gift from Feng Zhang (Addgene plasmid # 1765; ): pBABE-hygro vector was digested with BamHI and SalI and cDNA coding for human SLC1A3 (purchased from IDT) cloned using In-Fusion HD cloning kit from Clontech (Takara Bio USA, Inc).

Techniques: Activity Assay

Journal: Cell Metabolism

Article Title: A Role for p53 in the Adaptation to Glutamine Starvation through the Expression of SLC1A3

doi: 10.1016/j.cmet.2018.07.005

Figure Lengend Snippet: Deletion of SLC1A3 Impedes the ETC and Phenocopies Depletion of the Mitochondrial Aspartate Transporters AGC1 and AGC2 under Glutamine Deprivation (A) Schematic representation of the malate-aspartate shuttle (MAS). In brief, the MAS is a system that allows the transfer of electrons from cytosolic NADH to produce mitochondrial NADH where it is oxidized in the ETC. In the cytoplasm MDH1 catalyzes the reduction of oxaloacetate (OAA), where it accepts an electron from NADH to produce malate and NAD + . Malate can then enter the mitochondria where it is oxidized by MDH2 to OAA, resulting in the formation of mitochondrial NADH. Mitochondrial OAA is transaminated into aspartate by GOT2 whereby aspartate exits the mitochondria in exchange for cytosolic glutamate through a carrier. OAA is recovered in the cytosol by GOT1. By coupling aspartate-glutamate exchange, the aspartate-glutamate carrier is essential for the shuttle and is thought to represent the rate-limiting step. (B) Respiratory profiles of HCT116 WT cells transiently depleted of SLC1A3 and grown for 24 hr in glutamine-free medium, in the presence of mitochondrial inhibitors (oligomycin, FCCP [carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone] antimycin A/rotenone). Arrows indicate incubation of cells with the indicated inhibitors. Data are presented as mean ± SEM of one representative experiment (n = 6 wells). (C) Oxygen consumption rates (OCR) of HCT116 WT and p53-null clones 2 days after glutamine deprivation as in (B). Data are presented as mean ± SEM of one representative experiment (n = 6 wells). (D) Proliferation of HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA and cultured in glutamine-free condition (LHS) or complete medium (RHS). Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). The downregulation of these two proteins was confirmed by western blot (middle panel). (E) HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA were grown for 2 days in glutamine-free medium and pulsed with [U- 13 C]aspartate for the final 24 hr. Extracellular levels of aspartate (m+4), alanine, and serine normalized to cell number were quantified over 24 hr. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (F and G) HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA were grown for 2 days in glutamine-free medium and pulsed with [U- 13 C]aspartate for the final 24 hr. Intracellular TCA-cycle intermediates (F) and glutamate and glutamine levels (G) were measured. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). See also Figure S7 .

Article Snippet: Human SLC1A3 was cloned into the pBABE-hygro vector (gift from Feng Zhang (Addgene plasmid # 1765; ): pBABE-hygro vector was digested with BamHI and SalI and cDNA coding for human SLC1A3 (purchased from IDT) cloned using In-Fusion HD cloning kit from Clontech (Takara Bio USA, Inc).

Techniques: Incubation, Clone Assay, Cell Culture, Western Blot

Journal: Cell Metabolism

Article Title: A Role for p53 in the Adaptation to Glutamine Starvation through the Expression of SLC1A3

doi: 10.1016/j.cmet.2018.07.005

Figure Lengend Snippet: SLC1A3 Re-expression Rescues Survival and Metabolic Deficiency in p53-Null Cells under Glutamine Deprivation (A) p53-null HCT116 cells were infected with a control vector (EV) or a vector encoding SLC1A3 (SLC1A3). Western blot shows efficient SLC1A3 re-expression in these cells when cultured in complete medium (D0) or for 2 days in glutamine-free medium (D2). (B) Proliferation rates of the p53-null cells infected with a control vector or a vector encoding SLC1A3 under complete medium (LHS) or glutamine-free conditions (RHS). Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (C) Representative pictures of p53-null cells infected with a control vector or a vector encoding SLC1A3 and cultured for 3 days in glutamine-free medium. Viability of these cells grown for 4 days in glutamine-free medium was assessed by fluorescence-activated cell sorting. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (D) p53-null cells infected with a control vector or a vector encoding SLC1A3 were fed complete medium or glutamine-deficient medium for 2 days in the presence of [U- 13 C]aspartate for the final 16 hr. LC-MS was used for stable isotopomer tracing of TCA-cycle intermediates. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (E) Analysis of glutamate and glutamine levels in cells treated as in (D). Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (F) p53-null cells infected with a control vector or a vector encoding SLC1A3 were cultured for 3 days in complete medium in the presence of the glutaminase inhibitor CB-839 (0.1 μM, 0.5 μM, or 1 μM). The graph shows the percentage of growth compared with the untreated condition (DMSO). Data are presented as mean ± SEM from three independent experiments ( ∗ p < 0.05, ∗∗ p < 0.01, paired two-tailed Student’s t test).

Article Snippet: Human SLC1A3 was cloned into the pBABE-hygro vector (gift from Feng Zhang (Addgene plasmid # 1765; ): pBABE-hygro vector was digested with BamHI and SalI and cDNA coding for human SLC1A3 (purchased from IDT) cloned using In-Fusion HD cloning kit from Clontech (Takara Bio USA, Inc).

Techniques: Expressing, Infection, Plasmid Preparation, Western Blot, Cell Culture, Fluorescence, FACS, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: Cell Metabolism

Article Title: A Role for p53 in the Adaptation to Glutamine Starvation through the Expression of SLC1A3

doi: 10.1016/j.cmet.2018.07.005

Figure Lengend Snippet:

Article Snippet: Human SLC1A3 was cloned into the pBABE-hygro vector (gift from Feng Zhang (Addgene plasmid # 1765; ): pBABE-hygro vector was digested with BamHI and SalI and cDNA coding for human SLC1A3 (purchased from IDT) cloned using In-Fusion HD cloning kit from Clontech (Takara Bio USA, Inc).

Techniques: Recombinant, In Vitro, In Vivo, Clone Assay, Plasmid Preparation, Software, CRISPR