Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Serine 26 in Early Growth Response‐1 Is Critical for Endothelial Proliferation, Migration, and Network Formation

doi: 10.1161/JAHA.120.020521

Figure Lengend Snippet: A , CRISPR/Cas9 strategy to introduce Egr‐1 mutation. Using CRISPR/Cas9 single‐guide RNA (sgRNA) guide design platform ( http://crispr.mit.edu ), a guide was selected that gave a high score and where protospacer adjacent motif (PAM) silent mutation was possible. A target on the reverse strand (indicated by the thick black line) was chosen and annealed oligonucleotides cloned into the PX458 (digested with Bbs I). The generated PX‐458‐Egr‐1 was transfected into human microvascular endothelial cells together with the donor nucleotide to introduce a silent PAM mutation and point mutation of serine residue in Egr‐1 at position 26 (Ser26)>Ala. This CRISPR/Cas9 system generated indels by nonhomologous end joining and the desired mutation via homology‐directed repair (HDR). Transformants were sorted by fluorescence‐activated cell sorting, and single cells with green fluorescent protein were grown and later screened. The target sequence (blue), not including the PAM site (yellow), was inserted into the guide RNA (gRNA). sgRNA contains the custom‐designed sequence fused to the scaffold RNA sequence. Sequences of the target genomic DNA and donor oligonucleotide were shown. B , Alignment of amino acid sequences of different CRISPR/Cas9 clones with that of human Egr‐1 (P18146). Multiple sequence alignment with hierarchical clustering was done using MultAlin software ( http://multalin.toulouse.inra.fr/multalin/ ). C, In silico structure prediction by trRosetta of the amino‐terminal region of Egr‐1. The amino‐terminus of the protein starts at the helix. The first 63 amino acid residues of Egr‐1 were entered into trRosetta. Ser26 (yellow) is predicted to lie in a loop region between 2 helices. Wild‐type (WT) (with Ser26) and mutant (MUT) (Ala26) Egr‐1 sequence were run through the trRosetta program, and images were generated (using Pymol software). The WT model (shown in orange) was aligned structurally with the MUT model (blue). Residue 26 is highlighted in yellow. CVM indicates cytomegalovirus; Cas9, caspase 9; DEL, deletion; EGFP, enhanced green fluorescent protein; Egr‐1, early growth response‐1; and U6, the U6 promoter.

Article Snippet: The annealed oligonucleotide was ligated into the PX458/ Bbs I/CIP‐treated vector plasmid using the Quick T4 DNA ligase (New England Biolabs) and then transformed into XL‐10 Gold ultracompetent cells (Agilent, Santa Clara, CA).

Techniques: CRISPR, Introduce, Mutagenesis, Clone Assay, Generated, Transfection, Fluorescence, FACS, Sequencing, Software, In Silico

Journal: bioRxiv

Article Title: Single-cell analysis of bidirectional reprogramming between early embryonic states reveals mechanisms of differential lineage plasticities

doi: 10.1101/2023.03.28.534648

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The resulting PX458-E2-Crimson vector was digested using BbsI-Hf (NEB) and single guide RNA (sgRNA) targeting Gata4 or Gata6 was annealed as previously described ( ).

Techniques: Virus, Recombinant, SYBR Green Assay, Electron Microscopy, Immunoprecipitation, Reverse Transcription, Software

Journal: Journal of Cellular and Molecular Medicine

Article Title: Generation of ‘designer erythroblasts’ lacking one or more blood group systems from CRISPR/Cas9 gene‐edited human‐induced pluripotent stem cells

doi: 10.1111/jcmm.16872

Figure Lengend Snippet: CRISPR/Cas9 gene‐edited erythroblasts surface antigen presentation. Complete loss of RhAG, RhD/CE, GPB protein expression was confirmed for RhAG, RhD/CE (A) and GPB (B) erythroblasts. Anti‐U was used to confirm GPB deletion. Kell glycoprotein expression was evaluated as a proxy for XK knockout erythroblasts. Kell expression (C) was considerably diminished consistently with the lack of Kx expression. KO, knockout

Article Snippet: Briefly, cloning was performed by ligating annealed‐guides into BbsI digested GFP‐Cas9 expression vector pX458 (one guide/vector) using a quick ligation kit (New England Biolabs, Ipswich, MA, USA) and transformed into competent E. coli cells (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: CRISPR, Expressing, Knock-Out