Journal: Epigenetics

Article Title: Application of Recombination -Induced Tag Exchange (RITE) to study histone dynamics in human cells

doi: 10.1080/15592294.2020.1741777

Figure Lengend Snippet: Primers used in this study.

Article Snippet: Using identical transfection methods as described above, pX330 with gRNA 5ʹ-GGGGCCACTAGGGACAGGAT [ ] targeting the AAVS1 locus was co-transfected with an AAVS1 HDR plasmid (derived from AAVS1 hPGK-PuroR-pA donor, Hockemeyer et al. 2009 [ ]; Addgene #22072) containing a human PGK1 promoter driven Cre-ER T2 -IRES-NeoR.

Techniques: Sequencing

Journal: Epigenetics

Article Title: Application of Recombination -Induced Tag Exchange (RITE) to study histone dynamics in human cells

doi: 10.1080/15592294.2020.1741777

Figure Lengend Snippet: Strategy to knock-in a RITE cassette at H3F3B and Cre-ER T2 at the AAVS1 safe-harbour locus. (a) A RITE cassette (V5→FLAG) is integrated at the C-terminus of H3.3 ( H3F3B gene) using CRISPR-Cas9 and a repair plasmid for homologous recombination. Note that the gRNA is targeted to the last intron of H3F3B to prevent off-target cleavage at homologous histone H3 genes. (b) Genotyping PCR to confirm heterozygous integration of the RITE cassette at H3F3B in RPE1 and K562 cells. The asterisk denotes non-specific PCR amplicons. (c) A PGK1- promoter driven Cre-ER T2 is integrated at the AAVS1 safe-harbour locus using a HDR (homology-directed repair) plasmid donor derived from Mali et al. . (d) Genotyping PCR to confirm homozygous integration of Cre-ER T2 is at AAVS1 in RPE1 and K562 cells. The asterisk denotes non-specific PCR amplicons. HA, homology arm; PuroR-2A-EGFP, puromycin resistance gene followed by self-cleaving peptide P2A from porcine teschovirus-1 polyprotein and green fluorescent protein; IRES, internal ribosome entry site; NeoR, neomycin resistance gene.

Article Snippet: Using identical transfection methods as described above, pX330 with gRNA 5ʹ-GGGGCCACTAGGGACAGGAT [ ] targeting the AAVS1 locus was co-transfected with an AAVS1 HDR plasmid (derived from AAVS1 hPGK-PuroR-pA donor, Hockemeyer et al. 2009 [ ]; Addgene #22072) containing a human PGK1 promoter driven Cre-ER T2 -IRES-NeoR.

Techniques: Knock-In, CRISPR, Plasmid Preparation, Homologous Recombination, Derivative Assay

Journal: bioRxiv

Article Title: Comparative landscape of genetic dependencies in human and chimpanzee stem cells

doi: 10.1101/2023.03.19.533346

Figure Lengend Snippet:

Article Snippet: The CRISPRi effector protein dCas9-KRAB was introduced into either the CLYBL or AAVS1 safe harbor locus (Cheung et al., 1980; Hockemeyer et al., 2009; Philpott et al., 2002; Smith et al., 2008) via lipofection of three plasmids: 1) A modified version of pX458 (Addgene #48138), containing both Cas9 nuclease and a sgRNA targeting either CLYBL or AAVS1.

Techniques: Virus, Recombinant, Transfection, SYBR Green Assay, Flow Cytometry, Sequencing, Software