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. 2021 Sep 3;10(18):e020521. doi: 10.1161/JAHA.120.020521

Figure 1. Generation and validation of CRISPR/Cas9 clones.

Figure 1

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 BbsI). 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.