Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Suppression of female fertility in Aedes aegypti with a CRISPR-targeted male-sterile mutation

doi: 10.1073/pnas.2105075118

Figure Lengend Snippet: Generation of the B2t1 knock-in mutant and examination of testis-specific expression of B2t. (A) RT-PCR showing B2t mRNA expression in the indicated samples and sexes from wild type (WT) and B2t1. The locations of the primers used (F1 and R1) are illustrated in D. RpL32 is used as an internal control. The expected PCR products are 527 bp for B2t and 345 bp for RpL32. F, female; M, male; M-T, male without testis; T, testis. (B) Real-time RT-PCR showing relative B2t transcript levels in the indicated samples. The primer pair used (F2 and R2) is presented in D. Three biological replicates, each with three technical replicates, were used for each sample. Means ± SEMs. Statistics were performed using one-way ANOVA with the Tukey’s multiple comparisons test. n.s., not significant; ***P < 0.001. (C) Schematic of the pAaU6-LgRNA-3xP3-GFP plasmid used for engineering the knock-in constructs for CRISPR/Cas9-mediated genome editing of B2t. The PacI and NheI restriction sites were used to introduce the 5′ and 3′ homologous arms in B2t, respectively. The KpnI and SpeI restriction sites were used to clone the gRNA. The vector contains the following components: 3xP3-hsp70, three P3 binding elements and a minimal promoter from the Drosophila hsp70 gene; GFP, coding sequence for green fluorescent protein; SV40, SV40 transcriptional terminator; AaU6, Ae. aegypti U6 promoter; gRNA scaffold; attB sequence; ori, origin of replication; and ampR, ampicillin-resistant gene. (D) Schematic showing the 3xP3-GFP-SV40 knocked into the B2t gene to create the B2t1 allele. The location of the primers used in A, B, and E (F1, F2, R1, and R2) are indicated. (Scale bar, 200 bp.) (E) PCR genotyping of the B2t1 allele. The locations of the primers used are F1 and R1 and are presented in D. The expected PCR products are 584 bp in WT and 1.9 kb in B2t1. (F) Body sizes of WT and B2t1 mutant males. The image illustrates the area used for the length measurements. n = 4 to 5 groups. Each group included ≥5 mosquitoes. The total number of mosquitoes measured was 60 for WT and 32 for B2t1. Means ± SEMs are indicated. Statistics were performed using Mann–Whitney U test. n.s., not significant. (Scale bar, 500 μm.) (G) Wing lengths of WT and B2t1 mutant males. The image illustrates the area used for the length measurements. n = 5 groups. Each group included ≥10 wings. The total number of wings measured was 71 for WT and 101 for B2t1. Means ± SEMs are indicated. Statistics were performed using Mann–Whitney U test. n.s., not significant. (Scale bar, 500 μm.)

Article Snippet: We then subcloned this gRNA into the p AaU6-LgRNA-3xP3-GFP digested with KpnI and SpeI (the digestion removes the gRNA scaffold from the vector) using the In-Fusion Cloning system (Takara, In-Fusion HD Cloning Plus kit).

Techniques: Knock-In, Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Plasmid Preparation, Construct, CRISPR, Introduce, Binding Assay, Sequencing, MANN-WHITNEY

Journal: eNeuro

Article Title: Cell-Specific Single Viral Vector CRISPR/Cas9 Editing and Genetically Encoded Tool Delivery in the Central and Peripheral Nervous Systems

doi: 10.1523/ENEURO.0438-23.2024

Figure Lengend Snippet: Material availability

Article Snippet: Recombinant DNA reagent , pX552-EF1a-DIO-ChRonos-GFP(with gRNA scaffold) , This paper , Addgene #199582 , Expresses ChRonos-GFP in a Cre-dependent manner.

Techniques: Recombinant, Plasmid Preparation, Cloning, Control, Expressing, Sequencing, Software, CRISPR

Journal: eNeuro

Article Title: Cell-Specific Single Viral Vector CRISPR/Cas9 Editing and Genetically Encoded Tool Delivery in the Central and Peripheral Nervous Systems

doi: 10.1523/ENEURO.0438-23.2024

Figure Lengend Snippet: Grin1 knockdown selectively reduces NMDAR current in VTA DA neurons. A , Schematic of pX552 vector with Cre-dependent GCaMP8f transgene and Grin1 gRNA. B , Sequences of gRNA targeting Grin1 (top) and modified TTT control (bottom). C , Diagram of virus injection into the VTA of 6–8-week TH- Cre:Cas9 mice. D , Diagram of patch-clamp recording setup with recording electrode and stimulating electrode in the VTA. E , Representative traces from Grin1-edited neurons of evoked NMDAR current (top) and AMPAR current (bottom). Traces are aligned to stimulating electrode pulse. Each trace represents a single trial. Horizontal scale bar = 20 ms; vertical scale bar = 20 pA. F , Representative traces from TTT control neurons of evoked NMDAR current (top) and AMPAR current (bottom). Traces are aligned to stimulating electrode pulse. Each trace represents a single trial. Horizontal scale bar = 20 ms; vertical scale bar = 20 pA. G , Quantification of AMPAR EPSC amplitude in Grin1 (green, left) and TTT (gray, right) neurons. Each point represents the average peak AMPAR current amplitude from 10–15 consecutive sweeps for one neuron. The average Grin1 peak AMPAR current was −88.61 ± 7.39 pA ( n = 11 cells from 3 animals); the average TTT peak AMPAR current was −114.1 ± 11.47 pA ( n = 8 cells from 3 animals; p = 0.12; ns e ). H , Quantification of NMDAR EPSC amplitude in Grin1 (green, left) and TTT (gray, right) neurons. Each point represents the average peak NMDAR current amplitude from 10–15 consecutive sweeps for one neuron. The average Grin1 peak NMDAR current was 4.58 ± 1.41 pA ( n = 11 cells from 3 animals); the average TTT peak NMDAR current was 16.60 ± 1.90 pA ( n = 8 cells from 3 animals; p = 0.052; ns f ). I , Quantification of NMDAR:AMPAR ratio in Grin1 (green, left) and TTT (gray, right) neurons. Each point represents the ratio of the average peak NMDAR current amplitude to the average peak AMPAR current amplitude from 10–15 consecutive sweeps for one neuron. The average Grin1 NMDAR:AMPAR ratio was 0.053 ± 0.011 ( n = 11 cells from 3 animals); the average TTT NMDAR:AMPAR ratio was 0.146 ± 0.011 ( n = 8 cells from 3 animals; p = 0.027* g ). All comparisons were done using a two-tailed, nested t test. All data are reported as mean ± SEM. See Extended Data for more details.

Article Snippet: Recombinant DNA reagent , pX552-EF1a-DIO-ChRonos-GFP(with gRNA scaffold) , This paper , Addgene #199582 , Expresses ChRonos-GFP in a Cre-dependent manner.

Techniques: Knockdown, Plasmid Preparation, Modification, Control, Virus, Injection, Patch Clamp, Two Tailed Test