Journal: The Journal of Biological Chemistry

Article Title: Recharacterization of the mammalian cytosolic type 2 ( R )-β-hydroxybutyrate dehydrogenase as 4-oxo- l -proline reductase (EC 1.1.1.104)

doi: 10.1016/j.jbc.2022.101708

Figure Lengend Snippet: Western blot analysis of BDH2 expression in four Bdh2 -deficient HEK293T clonal cell lines ( Bdh2 -KO) and WT HEK293T cells (WT). The KO clonal cell lines (A12, A15, B9, and B13) were generated by the CRISPR–Cas9 gene-inactivation procedure. The Western blot analysis was carried out using 40 μg of the cell lysate protein, a primary rabbit antibody against the human BDH2 (catalog no.: PA5-44760; Invitrogen), and a horseradish peroxidase–conjugated goat anti-rabbit secondary antibody. The secondary antibody was detected by measuring enhanced chemiluminescence. The presence of a nonspecific signal (≈30 kDa) is in agreement with the specification of the primary antibody. BDH2, type 2 ( R )-β-hydroxybutyrate dehydrogenase; HEK293T, human embryonic kidney 293T cell line.

Article Snippet: This vector encodes an enhanced specificity Cas9 enzyme (a gift from Andrea Németh; Addgene; plasmid no.: 101039) ( ).

Techniques: Western Blot, Expressing, Generated, CRISPR

Journal: Microbial Biotechnology

Article Title: Developing a CRISPR‐assisted base‐editing system for genome engineering of Pseudomonas chlororaphis

doi: 10.1111/1751-7915.14075

Figure Lengend Snippet: Design of a CRISPR‐assisted base‐editing system tailored for Pseudomonas chlororaphis . A. Schematic illustration of the plasmid‐borne base‐editing system. The tool consists of an enhanced specificity Cas9 nickase (eSpCas9pp D10A ), fused to a cytidine deaminase (rAPOBEC1, which catalyses the C‐to‐T conversion), and the uracil DNA glycosylase inhibitor (UGI). The base‐editing machinery is directed to the target DNA by a single guide RNA (sgRNA). PAM , protospacer adjacent motif. B. Biochemical reaction catalysed by the cytidine deaminase rAPOBEC1. C. Structure of plasmid pBRC1, containing the construct that encodes the SpCas9pp D10A , rAPOBEC1 and UGI fusion. This module is under transcriptional control by the L‐arabinose‐inducible AraC/ P araBAD system, whereas the sgRNA is constitutively expressed from a P lac promoter. XTEN, short peptide linker sequence with no specific structure; N 20 , specific 20‐nt sequence targeting the gene to be edited; and Km R , kanamycin resistance. D. Strategy followed for gene inactivation by introducing premature STOP codons (TAA, TAG and TGA) in target genes using the base‐editing system. UGI inhibits uracil base excision on the non‐template DNA strand, so that DNA repair occurs on the template strand.

Article Snippet: A DNA cassette encoding the cytidine deaminase from rat [rAPOBEC1; Komor et al . ( )], an enhanced specificity Cas9 nickase [eSpCas9pp D10A , carrying the point mutations K848A, K1003A, R1060A and D10A; Sun et al . ( )], a XTEN linker [a short connector sequence; Komor et al . ( )] and the uracil DNA glycosylase inhibitor (UGI) was amplified from plasmid pSEVA2BE [a derivative of standard vector pSEVA644; Sun et al . ( )] and assembled in plasmid pBRC1 (deposited in Addgene, ID 183064).

Techniques: CRISPR, Plasmid Preparation, Construct, Control, Sequencing

Journal: European Journal of Endocrinology

Article Title: The phenotypic spectrum associated with OTX2 mutations in humans

doi: 10.1530/EJE-20-1453

Figure Lengend Snippet: OTX2 H230L had no abnormal phenotype, and C-terminal variants are tolerated. (A) CRISPR/Cas9 generated Otx2 H230L and multiple by-product variants. (B) Otx2 H230L had no obvious phenotype in either the pituitary gland or the eye. Otx2 L219fs*17 showed anophthalmia/microphthalmia but, however, showed similar pituitary morphology to WT animals. Arrows and arrowheads indicate the pituitary gland and optic nerve, respectively. (C) HE staining of eyes of adult mice. Otx2 H230L had normal eyes, but the retina of Otx2 L219fs*17 was thin. (D, E and F) Pituitary histology of P0 mice. (D) HE staining, (E) GH staining, and (F) LH staining were similar between genotypes.

Article Snippet: The Otx2 variant alleles were generated by microinjecting enhanced specificity Cas9 protein (30 ng/uL, Integrated DNA Technologies), DNA donor oligo (10 ng/µL, Integrated DNA Technologies), and C77G2 crRNA 10 ng/μL annealed with tracrRNA 15 ng/μL (Integrated DNA Technologies) into fertilized eggs obtained from super-ovulated B6CBAF1 females purchased from the Jackson Laboratory.

Techniques: CRISPR, Generated, Staining

Journal: bioRxiv

Article Title: Nonsense, but not frameshift, truncating mutations result in Cyclin D2 stabilisation in an induced pluripotent stem cell model of MPPH

doi: 10.1101/2025.08.29.673076

Figure Lengend Snippet: A - Electropherogram showing both alleles (top and bottom) of CCND2 biallelic frameshift line CCND2 FS-T1 compared to wildtype (middle) causing truncation at positions 266 and 272 on allele 1 and allele 2, respectively. B - illustrates the same but for the second CCND2 biallelic frameshift line CCND2 FS-T2 showing an 11 bp deletion on allele 1 (top) and a different 11 bp deletion on allele 2 (bottom) compared to wildtype (middle), causing CCND2 truncation at position 272 and 274 respectively. C - Sanger sequencing electropherograms indicating with arrows the single base pair change c.814G>T in heterozygous (top) and homozygous (bottom) CCND2 E272*Het and CCND2 E272*Hom CRISPR-Cas9 edited lines compared to wildtype (middle).

Article Snippet: A single-guide RNA targeting the final exon of CCND2 (5’-ACGTGACGGATCCAAGTCGG-3’) was ligated in to either the GFP-tagged CRISPR-Cas9 px458 plasmid (Addgene #48138) or the enhanced specificity Cas9 (eSpCas9, Addgene #71814) following digestion with BbsI restriction enzyme (Thermo Fisher Scientific).

Techniques: Sequencing, CRISPR

Journal: bioRxiv

Article Title: Nonsense, but not frameshift, truncating mutations result in Cyclin D2 stabilisation in an induced pluripotent stem cell model of MPPH

doi: 10.1101/2025.08.29.673076

Figure Lengend Snippet: A - 2D genomic and proteomic structure of CCND2, showing location of MPPH disease causing variants at the C-terminus (bottom) ( Mirzaa et al ., 2014 , Zhao et al ., 2024 ) and likely pathogenic variants from ClinVar in italics (top). Green indicates phosphorylation sites Ser269, Ser271 and Thr280, pink indicates ubiquitination site Lys270. B – Illustration of the CCND2 C- terminus showing the site of truncations, at the gene and protein level, in each CRISPR-Cas9 engineered line in comparison to wildtype (WT). Red hatched regions indicate regions of amino acid sequence changes due to a frameshift mutation. Green and pink highlighted regions signify locations of phosphorylation sites (Ser269, Ser271 and Thr280) and ubiquitination site Lys270, respectively.

Article Snippet: A single-guide RNA targeting the final exon of CCND2 (5’-ACGTGACGGATCCAAGTCGG-3’) was ligated in to either the GFP-tagged CRISPR-Cas9 px458 plasmid (Addgene #48138) or the enhanced specificity Cas9 (eSpCas9, Addgene #71814) following digestion with BbsI restriction enzyme (Thermo Fisher Scientific).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, CRISPR, Comparison, Sequencing, Mutagenesis

Journal: bioRxiv

Article Title: Nonsense, but not frameshift, truncating mutations result in Cyclin D2 stabilisation in an induced pluripotent stem cell model of MPPH

doi: 10.1101/2025.08.29.673076

Figure Lengend Snippet: Sections were stained for PAX6, β-Tubulin, TBR1, NeuN and Hoechst on all CCND2 CRISPR-Cas9 edited lines and compared to wildtype. For each, similar results were obtained from 2 independent batches grown. Scale bar 20um at x10 magnification.

Article Snippet: A single-guide RNA targeting the final exon of CCND2 (5’-ACGTGACGGATCCAAGTCGG-3’) was ligated in to either the GFP-tagged CRISPR-Cas9 px458 plasmid (Addgene #48138) or the enhanced specificity Cas9 (eSpCas9, Addgene #71814) following digestion with BbsI restriction enzyme (Thermo Fisher Scientific).

Techniques: Staining, CRISPR