cas9 nls purified protein  (Integrated DNA Technologies)

Journal: bioRxiv

Article Title: Genetic exploration of a nuclear receptor transcriptional regulatory complex

doi: 10.1101/2020.03.28.013060

Figure Lengend Snippet: (A) lir-1 and lin-26 genes are located downstream of lir-2 . NHR-25 ChIP-seq data (Thurtle-Schmidt et al., unpublished data) revealed NHR-25 occupied peak found in the long intron of lir-1 at about 3 kb upstream of the lir-1 / lin-26 operon. (B) The NHR-25 ChIP-seq peak sequence is highly conserved with other Caenorhabditis species (Multiz alignment, UCSC Genome Browser) and two NR5A-recognition consensus are found (boxed in blue) by JASPAR2020 (scan against NR5A2 matrix ID MA0505.1). (C) depicts NHR-25 ChIP-seq peak (blue bar), NR5A recognition elements (NR5ARE) are shown (light orange bars). Dark orange bars indicate legions in CRISPR-Cas9 generated deletion mutants (jm199, jm200 strains). (D) Brood size analyses of NR5ARE deletion mutant strains (left), CRISPR-Cas9 generated lir-2 mutants and original strain HL178 (right) at 20°C. 5-8 animals for each strain were scored. Standard error of the mean (SEM) is shown for each bar.

Article Snippet: Briefly, CRISPR-Cas9 ribonucleoprotein (RNP) complexes were preassembled by mixing 110 ng/μL tracr RNA (IDT), 100 ng/μL of each guide RNAs (Alt-R crRNA, IDT) and 250 ng/μL Cas9-NLS purified protein (MacroLab, QB3 Berkeley) and incubating at 37 °C for 10 min. 110 ng/μL of each ssODN donor templates (IDT) and 50 ng/μL pRF4: co-injection marker rol-6 (su1006) expression plasmid ( Kramer et al. 1990 ) were then added to the complex mixture.

Techniques: ChIP-sequencing, Sequencing, CRISPR, Generated, Mutagenesis

Journal: bioRxiv

Article Title: Genetic exploration of a nuclear receptor transcriptional regulatory complex

doi: 10.1101/2020.03.28.013060

Figure Lengend Snippet: (A) lir-2 genomic and protein structures are shown. Mutation S9 is indicated in red asterisk. Three LxxLL motifs are found at the position 161, 211 and 290 in LIR-2 and marked in green. The region containing LxxLL motifs (Lx1, Lx2 and Lx3) were aligned with ogous proteins in other Nematode species and showed that the region is highly conserved. LxxLL motifs icated over the amino acid sequences. (B) LIR-2 Protein structure of CRISPR-Cas9 generated mutants. dicates alteration in sequence, triangle indicates insertion, and deletion is marked as a white box. (C) sentative Venus expression in different tissues in mutants were shown. White bars in cr-178 and smo-1 panels indicate position of P5-7.pxx vulval cells. Scale bars: The black bar at the left top applies to all raphs of embryos and the black bar at the left bottom applies to all larval micrographs (50 μm).

Article Snippet: Briefly, CRISPR-Cas9 ribonucleoprotein (RNP) complexes were preassembled by mixing 110 ng/μL tracr RNA (IDT), 100 ng/μL of each guide RNAs (Alt-R crRNA, IDT) and 250 ng/μL Cas9-NLS purified protein (MacroLab, QB3 Berkeley) and incubating at 37 °C for 10 min. 110 ng/μL of each ssODN donor templates (IDT) and 50 ng/μL pRF4: co-injection marker rol-6 (su1006) expression plasmid ( Kramer et al. 1990 ) were then added to the complex mixture.

Techniques: Mutagenesis, CRISPR, Generated, Sequencing, Expressing

Journal: bioRxiv

Article Title: Genetic exploration of a nuclear receptor transcriptional regulatory complex

doi: 10.1101/2020.03.28.013060

Figure Lengend Snippet:

Article Snippet: Briefly, CRISPR-Cas9 ribonucleoprotein (RNP) complexes were preassembled by mixing 110 ng/μL tracr RNA (IDT), 100 ng/μL of each guide RNAs (Alt-R crRNA, IDT) and 250 ng/μL Cas9-NLS purified protein (MacroLab, QB3 Berkeley) and incubating at 37 °C for 10 min. 110 ng/μL of each ssODN donor templates (IDT) and 50 ng/μL pRF4: co-injection marker rol-6 (su1006) expression plasmid ( Kramer et al. 1990 ) were then added to the complex mixture.

Techniques: CRISPR

Journal: bioRxiv

Article Title: Spinal neuron diversity scales exponentially with swim-to-limb transformation during frog metamorphosis

doi: 10.1101/2024.09.20.614050

Figure Lengend Snippet: A-B. Generation of unilateral FoxP1 CRISPR mutant frogs by injection of FoxP1 sgRNA and Cas9 protein in a single cell at two-cell stage ( A ) results in NF54-55 tadpoles in which FoxP1 (red) and Raldh2 (green) immunoreactivity is selectively absent from the mutant side of the spinal cord ( B ). Isl1/2-positive (blue, marker for motor neurons in ventral spinal cord) neurons are present on both wildtype and mutant side of spinal cord ( B ). Scale bar, 50 μm. C-D. Cell-type characterization in unilateral FoxP1 CRISPR mutant animals. Quantification of spinal cord cell numbers at brachial (Br), thoracic (Th) and lumbar (Lu) reveals loss of FoxP1+ Isl1+ neurons at all levels ( C ; uninjected vs. FoxP1 ½: Br, p = <0.0001; for Th and Lu, p = 0.002) and loss of Raldh2+ Isl1+ neurons at brachial and lumbar levels ( D ; uninjected vs. FoxP1 ½: Br, p = 0.025; Lu, p = 0.029). n = 6 for WT, n = 6 unilateral FoxP1 CRISPR. E-I. Loss of range and coordination of movement of the FoxP1 mutant hindlimb. WT ( E ) and unilateral FoxP1 CRISPR ( F ) juvenile frogs with SLEAP skeleton (left, yellow) superimposed on animal image. PCA plots represent the position of the fore and hind limb and their range of movement during 256 random frames and show a different position and range of the FoxP1 CRISPR limbs compared to WT or the uninjected side ( E-F , right; hip and shoulder, yellow; knee and elbow, orange; ankle and wrist, red; foot, brown). Scale bar in E and F indicates the color-code of the first principal component of variation of the aligned fore and hind limb positions. The FoxP1 CRISPR mutant knee also differs in its mean angle ( G ; for WT L versus FoxP1 ½, WT R versus FoxP1 ½ and uninjected versus FoxP1 ½, p = <0.0001), and its movement range is reduced ( I ; WT L versus FoxP1 ½, p = 0.0006; WT R versus FoxP1 half, p = 0.0002; uninjected versus FoxP1 1/2, p = <0.0001). In contrast, the uninjected side displays a higher range of movement ( H ; WT L versus uninjected, p = 0.009; WT R versus uninjected, p = 0.025). Left-right coordination between knee joints is lost in FoxP1 CRISPR animals ( I ; +1 = bilateral synchronous, 0 = random, -1 = alternate synchronous; WT versus FoxP1 ½ CRISPR, p = <0.0001). n = 13 for WT, n = 14 for unilateral FoxP1 CRISPR. J-L. FoxP1 CRISPR mutant hindlimbs maintain dominant frequency but lose power. Mean power spectrum of knee oscillations shows only one peak in the low frequency range for WT, uninjected and FoxP1 CRISPR hindlimbs ( J ; 0.9-4.5 Hz, dark gray). At the knee joint, the amount of movement in the low frequency bin (0.9-4.5 Hz), represented by the sum power, is lower on the mutant side compared to both the uninjected side and WT ( K ; for WT L versus FoxP1 ½ CRISPR and WT R versus FoxP1 ½ CRISPR, p = <0.0001; uninjected versus FoxP1 ½ CRISPR, p = 0.021). Dominant frequency is unaffected on both uninjected and FoxP1 CRISPR sides ( L ). n = 13 for WT, n = 14 for unilateral FoxP1 CRISPR.

Article Snippet: Embryos were injected with either 3 or 16 ng of purified Cas9-NLS protein diluted in DNA-free water (Synthego), and 5 ng sgRNA (Synthego) either (i) twice into diagonally opposite points of the animal pole between 20 minutes and before cleavage to generate bilateral mutant animals, or (ii) once into only one cell at two-cell stage to generate unilateral mutant animals.

Techniques: CRISPR, Mutagenesis, Injection, Marker

Journal: bioRxiv

Article Title: Spinal neuron diversity scales exponentially with swim-to-limb transformation during frog metamorphosis

doi: 10.1101/2024.09.20.614050

Figure Lengend Snippet: A-B. Generation of bilateral FoxP1 CRISPR mutant frogs. Injection of FoxP1 sgRNA and Cas9 protein at one cell stage ( A ). Resulting mutants ( B right) largely lacked FoxP1 (red) and Raldh2 (green) immunoreactivity as compared to wiltype ( B left). Isl1/2-positive (blue) motor neurons were present in both conditions ( B ). C-D . Quantification of bilateral FoxP1 mutants showed that FoxP1 + ( C ) and Raldh2 + (LMC, D ) motor neurons were decreased at all spinal levels at NF54 (p < 0.05 for all levels except for Raldh2 at thoracic levels). Shown are mean ± SEM (n = 6 animals) per 15 μm ventral horn. E-F. Genomic characterization of unilateral and bilateral FoxP1 CRISPR mutant animals. TIDE analysis reveals high efficiency of FoxP1 sgRNA in generating bilateral mutant animals at NF44-48 ( E ; WT vs mutant, p = 0.024, n = 3 for WT and n = 6 for FoxP1 animals), as well as unilateral mutant animals at juvenile stage ( F ; n = 2 for WT and n = 4 for FoxP1 animals). G-J. Profiling of other spinal neuron types in FoxP1 mutant. Quantification of MMC (Hb9 + Lhx3 + , G ) and V1 (En1 + , H ) neurons on the mutant and uninjected side of the spinal cord at all spinal levels. Shown are mean ± SEM (n = 3–6 animals) per 15 μm ventral horn V1 1TF ( I ) and V1 2TF ( J ) subtypes are largely unaffected in unilateral FoxP1 mutants at NF54-55. Shown are mean ± SEM (n = 3–6 animals) per 15 μm ventral horn. K-L. Retrograde labeling with rhodamine dextran (RhD, red) labels LMC motor neurons (FoxP1+, blue; Raldh2+, green; Isl1/2+, white) that project to the hindlimb in both wildtype (L ) and unilateral FoxP1 CRISPR ( M ) mutant animals. Scale bar, 40 μm.

Article Snippet: Embryos were injected with either 3 or 16 ng of purified Cas9-NLS protein diluted in DNA-free water (Synthego), and 5 ng sgRNA (Synthego) either (i) twice into diagonally opposite points of the animal pole between 20 minutes and before cleavage to generate bilateral mutant animals, or (ii) once into only one cell at two-cell stage to generate unilateral mutant animals.

Techniques: CRISPR, Mutagenesis, Injection, Labeling

Journal: bioRxiv

Article Title: Spinal neuron diversity scales exponentially with swim-to-limb transformation during frog metamorphosis

doi: 10.1101/2024.09.20.614050

Figure Lengend Snippet: A-C. Characterization of En1 CRISPR mutant animals. En1 sgRNA and Cas9 protein were injected at one cell stage to generate bilateral mutant animals, resulting in loss of En1 immunoreactivity from both sides of the spinal cord at NF39-40 ( A right, white), or in one cell at two-cell stage to generate unilateral mutant animals with loss of En1 immunoreactivity only from the En1 CRISPR side of the spinal cord at NF54-55 ( B right, white). TIDE analysis reveals high efficiency of En1 sgRNA in generating NF44-48 bilateral, and ∼25% mutation rate for juvenile unilateral CRISPR animals ( C ; n = 8 for WT, n = 36 for En1 bilateral mutants, n = 8 for En1 unilateral mutants). Scale bar, 50 μm. D-H. Range and coordination of movement are unaffected in juvenile unilateral En1 CRISPR mutant animals. WT ( D ) and unilateral En1 CRISPR ( E ) juvenile frogs with SLEAP skeleton (yellow) superimposed on animal image. PCA plots represent the position of the fore and hind limb and their range of movement during 256 random frames and show no visible difference in range between WT, uninjected and En1 CRISPR sides ( D-E, right; hip and shoulder, yellow; knee and elbow, orange; ankle and wrist, red; foot, brown). Scale bar in D and E indicates the color-code of the first principal component of variation of the aligned fore and hind limb positions. Unilateral En1 CRISPR mutant knees show similar mean angle ( F ) and angle range ( G ) as WT. Left-right coordination between knee joints is also unaffected in unilateral En1 CRISPR animals, as their pattern of movement resembles the bilateral synchronicity of WT ( H ; +1 = bilateral synchronous, 0 = random, -1 = alternate synchronous). n = 13 for WT, n = 8 for unilateral En1 CRISPR. I-K. Lower dominant frequency in juvenile En1 CRISPR mutant hindlimbs. Mean power spectrum of the knee oscillation shows only one peak in the low frequency bin (0.9-4.5 Hz, dark gray) for WT and unilateral En1 CRISPR animals ( I ). At the knee joints, the amount of movement, represented by the sum of the power, is similar between WT and unilateral En1 CRISPR animals ( J ). However, the dominant frequency of the knees is lower in unilateral En1 CRISPR animals compared to WT ( K ; WT vs En1 ½ CRISPR, p = 0.0002). n = 13 for WT, n = 8 for unilateral En1 CRISPR.

Article Snippet: Embryos were injected with either 3 or 16 ng of purified Cas9-NLS protein diluted in DNA-free water (Synthego), and 5 ng sgRNA (Synthego) either (i) twice into diagonally opposite points of the animal pole between 20 minutes and before cleavage to generate bilateral mutant animals, or (ii) once into only one cell at two-cell stage to generate unilateral mutant animals.

Techniques: CRISPR, Mutagenesis, Injection

Journal: iScience

Article Title: A lipid transfer protein ensures nematode cuticular impermeability

doi: 10.1016/j.isci.2022.105357

Figure Lengend Snippet:

Article Snippet: Cas9-NLS Purified Protein , IDT-DNA , Cat# 1081058.

Techniques: Virus, Recombinant, Purification, CRISPR, Knock-In, Plasmid Preparation, Modification, Expressing, Software