Feehan JM , Chiu CN , Stanar P , Tam BM , Ahmed SN , Moritz OL .

             retinal rod cell development

Xla Wt + rho CRISPR (Fig.1.B)
Xla Wt + rho CRISPR (Fig.2.A)
Xla Wt + rho CRISPR (Fig.2.A)
Xla Wt + rho CRISPR (Fig.2.B)
Xla Wt + rho CRISPR (Fig2.B)

	Figure 1. Injection of Cas9 and guide RNAs causes reduced rod opsin levels and retinal degeneration. (A) Rod opsin levels assayed in individual eyes by dot blot assay at 14 dpf. Each data point represents a different animal. P values are for Tukey multiple comparisons test for comparisons with WT group. P (ANOVA) = 4.1 × 10−19 (B) Phenotypes assessed by confocal microscopy. Retinas from animals injected with Cas9 mRNA and rhosg3 had missing and malformed rod photoreceptors. Green: anti-rhodopsin (B630N). Red: wheat germ agglutinin. Blue: Hoechst 33342. RPE: retinal pigment epithelium. OS: outer segments. IS: inner segments. ONL: outer nuclear layer. INL: inner nuclear layer. Bar = 50 µm.
	Figure 2. Relative to guide RNA rhosg3, rhosg1 causes greater reductions in rod opsin and greater rod photoreceptor loss. 6 ng of Cas9 mRNA and 2 ng of sgRNA were co-injected. (A) Rod opsin levels assayed in individual eyes by dot blot assay at 14 dpf. Each data point represents a different animal. P values are for Tukey multiple comparisons test for comparisons with WT group. P (ANOVA) = 2.9 × 10−9 (B) Phenotypes assessed by confocal microscopy. Green: anti-rhodopsin (B630N). Red: wheat germ agglutinin. Blue: Hoechst 33342. RPE: retinal pigment epithelium. OS: outer segments. IS: inner segments. ONL: outer nuclear layer. INL: inner nuclear layer. Bar = 50 µm.
	Figure 3. Indels are present in the genomic DNA of F0 animals. (A,B) Sample trace reads from dye-termination Sanger sequencing. (A) WT sample. (B) Edited with rhosg1. Cleavage is predicted to occur after the nucleotide highlighted in blue (numbered “zero” on plots below). (C–F) Comparisons of gene editing efficiency between guide RNAs, genes, and stage of development. Calculation of the sequence fidelity score is described under Methods. Each plot shows data derived from three animals, numbered 1–3, 4–6, 7–9 and 10–12.
	Figure 4. Characterization of indels by sequencing genomic DNA clones: (A–H) Sequences obtained from individual pBluescript-SKII+ clones of PCR products derived from genomic DNA isolated from F0 embryos at either 1 dpf or 14 dpf (as indicated) edited with Cas9 mRNA+ rhosg1 or rhosg3 (as indicated). The reverse complement sequence of the guide RNA is shown above the individual genomic DNA sequences in blue. The targeted sequences are also shown in blue. Mismatches with the guide RNA are shown in magenta. Insertions are noted in green. The protospacer adjacent motif (PAM) sequence is shown in red. Bases that are diagnostic of rho.L vs. rho.2.L are shown in orange. The number at the beginning of each sequence corresponds to sample numbers for direct sequencing results shown in Fig. 3.
	Figure 5. Germline transmission of genomic DNA editing and phenotypes of F1 offspring: Plots: Rod opsin levels assayed in individual eyes by dot blot assay at 14 dpf. Each data point represents a different F1 animal. Each X-axis point represents offspring of a single mating, for which the F0 parents are indicated as being WT and/or F0 animals (named Male 1, Male 2, etc.). Each plot represents samples analyzed on a separate dot blot, and all animals on each plot were modified using the same sgRNA, indicated at the bottom of the plot. (A–I) Phenotypes of F1 animals assessed by confocal microscopy. (A) Wildtype. (B–I) genetically modified (genotypes indicated on panels, for frame-conserving mutations the altered amino acid sequence is shown, with inserted amino acid residues shown in red). (B,C) frame-conserving mutations with minimal phenotype. (D–F) frame-conserving indels with significant RD phenotypes. (G–I) frame shifting indels. (A–C) High magnification panels: frame-conserving indels generating minimal or no RD phenotype do not alter rhodopsin localization, which is largely confined to outer segments (OS) and wheat germ agglutinin-positive inner segment (IS) membranes (arrowheads). (D–F) High magnification panels: frame-conserving indels generating significant RD phenotypes alter rhodopsin localization in inner segments causing a punctate distribution (D, arrowhead) or diffuse labeling consistent with ER retention (E,F, arrowheads). Green: anti-rhodopsin (B630N – A-I or 514–18 A-D high mag). Red: anti-rod transducin. Blue: Hoechst 33342. Bars = 20 µm (low mag) or 5 µm (high mag). RPE: retinal pigment epithelium. OS: outer segments. IS: inner segments. ONL: outer nuclear layer. INL: inner nuclear layer.
	Figure 6. Electron microscopy of rod photoreceptors with loss of function mutations in one rho.L allele: (A) images of rods with a 1 bp insertion in one rho.L allele induced by rhosg3. (B) Comparable images from WT rods. Outer segment (OS) disks are densely packed at both the rim and lamellar (LAM) regions. Disk rims are closely apposed to the plasma membrane (PM). No significant abnormalities were identifiable. IS: inner segment. Bar = 400 nm (left) 200 nm (center) or 100 nm (right).
	Figure 7. HDR mediated gene alterations assessed by confocal microscopy: (A) RD in an animal edited using sgRNA rhosg4 (B,C,C’) images derived from an animal in which rhosg4 was used to direct targeted insertion of eGFP into rho.L. A number of eGFP-positive rods are present, while other cells of the retina and lens do not show eGFP expression. Significant RD is present (D,E) images derived from an animal with targeted mutation of residue M13→F in rho.L, and stained with anti-mammalian rhodopsin (2B2). There is no identifiable RD. (A–C) Green: eGFP. (D,E) green: anti-mammalian rhodopsin (2B2). (A,B) Red: anti-rod transducin. C–E: Red: wheat germ agglutinin. (A–E) Blue: Hoechst 33342. OS: outer segments. IS: inner segments. ONL: outer nuclear layer. INL: inner nuclear layer. GCL: ganglion cell layer. (A,B,D) Bar = 200 µm. (C) Bar = 50 µm. (C’) Bar = 10 µm. (E) Bar = 20 µm. Panels C and C’ are confocal projections derived from 10 confocal sections.

Allen, Transgenic Xenopus laevis embryos can be generated using phiC31 integrase. 2005, Pubmed, Xenbase

Allen, Transgenic Xenopus laevis embryos can be generated using phiC31 integrase. 2005, Pubmed , Xenbase
Batni, Characterization of the Xenopus rhodopsin gene. 1996, Pubmed , Xenbase
Bell, A simple way to treat PCR products prior to sequencing using ExoSAP-IT. 2008, Pubmed
Bogéa, Light Induces Ultrastructural Changes in Rod Outer and Inner Segments, Including Autophagy, in a Transgenic Xenopus laevis P23H Rhodopsin Model of Retinitis Pigmentosa. 2015, Pubmed , Xenbase
Callery, There's more than one frog in the pond: a survey of the Amphibia and their contributions to developmental biology. 2006, Pubmed , Xenbase
Chen, Co-expression of Argonaute2 Enhances Short Hairpin RNA-induced RNA Interference in Xenopus CNS Neurons In Vivo. 2009, Pubmed , Xenbase
Chen, High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. 2011, Pubmed
Chesneau, Transgenesis procedures in Xenopus. 2008, Pubmed , Xenbase
Chu, Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. 2015, Pubmed
Cong, Multiplex genome engineering using CRISPR/Cas systems. 2013, Pubmed
Evans, Genome evolution and speciation genetics of clawed frogs (Xenopus and Silurana). 2008, Pubmed , Xenbase
Gauss, Mechanistic constraints on diversity in human V(D)J recombination. 1996, Pubmed
Gibson, Enzymatic assembly of DNA molecules up to several hundred kilobases. 2009, Pubmed
Gross, Defective development of photoreceptor membranes in a mouse model of recessive retinal degeneration. 2006, Pubmed
Heasman, Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. 2000, Pubmed , Xenbase
Hicks, Differential immunogold-dextran labeling of bovine and frog rod and cone cells using monoclonal antibodies against bovine rhodopsin. 1986, Pubmed , Xenbase
Hsu, Development and applications of CRISPR-Cas9 for genome engineering. 2014, Pubmed
Humphries, Retinopathy induced in mice by targeted disruption of the rhodopsin gene. 1997, Pubmed
Hwang, Efficient genome editing in zebrafish using a CRISPR-Cas system. 2013, Pubmed
Jin, An improved rhodopsin/EGFP fusion protein for use in the generation of transgenic Xenopus laevis. 2003, Pubmed , Xenbase
Jin, Opsin activation as a cause of congenital night blindness. 2003, Pubmed , Xenbase
Jinek, A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. 2012, Pubmed
Karpinka, Xenbase, the Xenopus model organism database; new virtualized system, data types and genomes. 2015, Pubmed , Xenbase
Knight, Dynamics of CRISPR-Cas9 genome interrogation in living cells. 2015, Pubmed
Kroll, Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. 1996, Pubmed , Xenbase
Lee, Dysmorphic photoreceptors in a P23H mutant rhodopsin model of retinitis pigmentosa are metabolically active and capable of regenerating to reverse retinal degeneration. 2012, Pubmed , Xenbase
Lem, Morphological, physiological, and biochemical changes in rhodopsin knockout mice. 1999, Pubmed
Lieber, The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. 2010, Pubmed
LIEBMAN, In situ microspectrophotometric studies on the pigments of single retinal rods. 1962, Pubmed
Lin, IRE1 signaling affects cell fate during the unfolded protein response. 2007, Pubmed
Lobanova, Proteasome overload is a common stress factor in multiple forms of inherited retinal degeneration. 2013, Pubmed
Makino, Rhodopsin expression level affects rod outer segment morphology and photoresponse kinetics. 2012, Pubmed
Mali, RNA-guided human genome engineering via Cas9. 2013, Pubmed
Maruyama, Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. 2015, Pubmed
Matsuda, A New Nomenclature of Xenopus laevis Chromosomes Based on the Phylogenetic Relationship to Silurana/Xenopus tropicalis. 2015, Pubmed , Xenbase
Moritz, A functional rhodopsin-green fluorescent protein fusion protein localizes correctly in transgenic Xenopus laevis retinal rods and is expressed in a time-dependent pattern. 2001, Pubmed , Xenbase
Moritz, Mutant rab8 Impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus rods. 2001, Pubmed , Xenbase
Moritz, Recent insights into the mechanisms underlying light-dependent retinal degeneration from X. laevis models of retinitis pigmentosa. 2010, Pubmed , Xenbase
Nakayama, Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis. 2013, Pubmed , Xenbase
Nutt, Comparison of morpholino based translational inhibition during the development of Xenopus laevis and Xenopus tropicalis. 2001, Pubmed , Xenbase
Ogino, High-throughput transgenesis in Xenopus using I-SceI meganuclease. 2006, Pubmed , Xenbase
Ohnuma, Lipofection strategy for the study of Xenopus retinal development. 2002, Pubmed , Xenbase
Pearl, Development of Xenopus resource centers: the National Xenopus Resource and the European Xenopus Resource Center. 2012, Pubmed , Xenbase
Price, Rhodopsin gene expression determines rod outer segment size and rod cell resistance to a dominant-negative neurodegeneration mutant. 2012, Pubmed
Rosenfeld, A null mutation in the rhodopsin gene causes rod photoreceptor dysfunction and autosomal recessive retinitis pigmentosa. 1992, Pubmed
San Filippo, Mechanism of eukaryotic homologous recombination. 2008, Pubmed
Schröder, The mammalian unfolded protein response. 2005, Pubmed
Schulte-Merker, Out with the old, in with the new: reassessing morpholino knockdowns in light of genome editing technology. 2014, Pubmed
Session, Genome evolution in the allotetraploid frog Xenopus laevis. 2016, Pubmed , Xenbase
Tam, Mislocalized rhodopsin does not require activation to cause retinal degeneration and neurite outgrowth in Xenopus laevis. 2006, Pubmed , Xenbase
Tam, The role of rhodopsin glycosylation in protein folding, trafficking, and light-sensitive retinal degeneration. 2009, Pubmed , Xenbase
Tam, The C terminus of peripherin/rds participates in rod outer segment targeting and alignment of disk incisures. 2004, Pubmed , Xenbase
Tam, Photoactivation-induced instability of rhodopsin mutants T4K and T17M in rod outer segments underlies retinal degeneration in X. laevis transgenic models of retinitis pigmentosa. 2014, Pubmed , Xenbase
Tam, Characterization of rhodopsin P23H-induced retinal degeneration in a Xenopus laevis model of retinitis pigmentosa. 2006, Pubmed , Xenbase
Tam, Generation of transgenic X. laevis models of retinal degeneration. 2013, Pubmed , Xenbase
Tam, Dark rearing rescues P23H rhodopsin-induced retinal degeneration in a transgenic Xenopus laevis model of retinitis pigmentosa: a chromophore-dependent mechanism characterized by production of N-terminally truncated mutant rhodopsin. 2007, Pubmed , Xenbase
Tam, Identification of an outer segment targeting signal in the COOH terminus of rhodopsin using transgenic Xenopus laevis. 2000, Pubmed , Xenbase
Tam, The dependence of retinal degeneration caused by the rhodopsin P23H mutation on light exposure and vitamin a deprivation. 2010, Pubmed , Xenbase
Tam, Preparation of Xenopus laevis retinal cryosections for electron microscopy. 2015, Pubmed , Xenbase
Tandon, Expanding the genetic toolkit in Xenopus: Approaches and opportunities for human disease modeling. 2017, Pubmed , Xenbase
Vouillot, Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. 2015, Pubmed , Xenbase
Wang, Targeted gene disruption in Xenopus laevis using CRISPR/Cas9. 2015, Pubmed , Xenbase
Wang, One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. 2013, Pubmed
Wen, Overexpression of rhodopsin alters the structure and photoresponse of rod photoreceptors. 2009, Pubmed
Yang, One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. 2013, Pubmed