human codon optimized streptococcus pyogenes cas9 gene  (Addgene inc)

Journal: Nature Communications

Article Title: CoCas9 is a compact nuclease from the human microbiome for efficient and precise genome editing

doi: 10.1038/s41467-024-47800-9

Figure Lengend Snippet: a Phylogenetic tree of selected Cas9 subfamilies mostly used for genome editing applications (blue) or with experimentally determined three-dimensional structure (orange). Protein alignments: gray, aligned protein sequences; black, conserved residues. Length: number of amino acids. b Scheme of the CRISPR locus where CoCas9 is located. c tracrRNA and crRNA predicted secondary structure. d Sequence logo representation of the PAM of CoCas9, determined using an in vitro cleavage assay. e Heatmap showing relative frequency of the 256 PAM nucleotide combinations at the four most informative positions, compared to the non-cleaved PAM library. Source data are provided as a Source Data file.

Article Snippet: In brief: the synthetic DNA encoding the human codon optimized version of the Cas9 genes was obtained from Genscript and cloned into an expression vector for in vitro transcription and translation (IVT) (pT7-N-His-GST Thermo Fisher Scientific).

Techniques: CRISPR, Sequencing, In Vitro, Cleavage Assay

Journal: Microbial cell factories

Article Title: SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae.

doi: 10.1186/s12934-017-0632-x

Figure Lengend Snippet: Fig. 2 Efficient setup for SWITCH. a Screening for gRNAs that effi- ciently targets Cas9 to the cas9 gene, using TAPE, see text for details. b Confirmation of X-3 locus restoration by genomic PCR of 12 clones randomly selected amongst the transformants obtained in the pres- ence of a X-3 rescue fragment. The presence of a 1.5 kb PCR fragment indicates that the X-3 locus has been restored

Article Snippet: To generate the different types of cas9 gene fragments for integration into the X-3 locus, the human codon optimized cas9 gene from Addgene plasmid #43802 and the yeast codon optimized dcas9 from Addgene plasmid #64279, were PCR amplified using USER compatible primers.

Techniques: Clone Assay

Journal: Microbial cell factories

Article Title: SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae.

doi: 10.1186/s12934-017-0632-x

Figure Lengend Snippet: Fig. 4 By SWITCH, the cas9 gene can be efficiently replaced by dcas9 and dcas9_VP64 genes. a dcas9 and dcas9_VP64 fragments efficiently rescue DNA DSBs formed by Cas9-gRNA_16 in the cas9 gene. b Confirmation of SWITCH gene replacements by PCR of 12 randomly selected clones co-transformed with gRNA_16 and (top) the dcas9 rescue fragment and (bottom) the dcas9_VP64 fragment. Diagnos- tic PCR fragments for dcas9 and dcas9_VP64 are 821 and 977 bp, respectively

Article Snippet: To generate the different types of cas9 gene fragments for integration into the X-3 locus, the human codon optimized cas9 gene from Addgene plasmid #43802 and the yeast codon optimized dcas9 from Addgene plasmid #64279, were PCR amplified using USER compatible primers.

Techniques: Clone Assay, Transformation Assay

Journal: Microbial cell factories

Article Title: SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae.

doi: 10.1186/s12934-017-0632-x

Figure Lengend Snippet: Fig. 5 Implementing and exploiting the regulatory state of SWITCH. a Localization of the 15 gRNAs tested for GAL2 promoter activation. b Screen- ing of the efficiency of the 15 gRNAs to guide Cas9 to the GAL2 promoter using TAPE. c Evaluation of ADE2 expression in colony patches on both SC-Ade-Leu and SC-Leu solid media. d Determination of ADE2 transcript levels relative to ACT1 by qRT-PCR in selected strains as indicated. Stars above columns indicate strains with an ADE2 expression level, which is significantly different (p values <0.005) from the corresponding level obtained from a control strain harboring pRS415

Article Snippet: To generate the different types of cas9 gene fragments for integration into the X-3 locus, the human codon optimized cas9 gene from Addgene plasmid #43802 and the yeast codon optimized dcas9 from Addgene plasmid #64279, were PCR amplified using USER compatible primers.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Control