Journal: Nature Communications

Article Title: Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9

doi: 10.1038/s41467-021-25991-9

Figure Lengend Snippet: a The sequence of the lentiviral gRNAHNF4A and its PAM site in blue. Above is the reference sequence of the HNF4A gene near the gRNA target site, as validated by Sanger sequencing in primary human hepatocytes expressing via lentivirus Cas9 and gRNAscr, with the TSS indicated by a black arrow and the reference protein sequence in red. CGs are bolded and underlined. Below is the dominant Sanger sequence profile of a primary human hepatocyte population expressing lentiviral Cas9 and gRNAHNF4A. This mutation and the resulting difference in the amino acid sequence, as well as the reference sequences at this location, are highlighted in yellow. b Two technical replicates each of the Sanger sequencing chromatograms from the primary human hepatocytes expressing dCas9 and gRNAscr (left) or dCas9 and gRNAHNF4A (right) at the targeted HNF4A locus. c Sanger sequencing results of 13 gRNAscr and 12 gRNAHNF4A DNA strands following bisulfite conversion from the cell populations in ( b ), demonstrating both the methylation levels and the variety of mutations induced by Cas9 in gHNF4A-treated cells. d Same as ( c ) except data expanded is expanded to a larger (>300 bp) region, and simplified such that only CpGs are shown, where blue squares indicate unmethylated CpGs, red squares indicate methylated CpGs, and white squares indicate missing information due to Cas9-induced deletions. CpGs are numbered in accordance with ( a ). e Bisulfite-sequencing data from ( d ) (center) as well as five CpGs immediately upstream (left) and seven CpGs immediately downstream (right), displayed as percent DNA methylation over all sequenced DNA strands in primary human hepatocytes expressing Cas9 and either gRNAscr (gray) or gRNAHNF4A (orange) and as mean ± SD as it is summary data from one mutated cell line. Individual dots represent individual strands of DNA from this clonal cell line. f HNF4A expression in primary human hepatocytes expressing Cas9 and either gRNAscr (gray) or gRNAHNF4A (orange) quantified by RT-qPCR and normalized to GAPDH expression, followed by normalization to average expression in gRNAscr cells, with a dashed line at 1 ( n = 6 independent clones, mean ± SD). * indicates statistically significant difference of P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns not significant (Student’s t -test, two-sided, with Holm-Sidak correction if number of tests is greater than 3). Source data are provided as a Source Data file.

Article Snippet: The HNF4A -targeting gRNA is from the genome-scale CRISPR knock-out (GeCKO) v2 library (purchased as lentiviral plasmid from Genscript) and the FMR1-targeting gRNA from the Jaenisch lab was obtained from Addgene (pgRNA-CGG, Addgene #108248).

Techniques: Sequencing, Expressing, Mutagenesis, Methylation, Methylation Sequencing, DNA Methylation Assay, Quantitative RT-PCR, Clone Assay

Journal: Journal of Fungi

Article Title: Comparison of CRISPR-MAD7 and CRISPR-Cas9 for Gene Disruptions in Komagataella phaffii

doi: 10.3390/jof10030197

Figure Lengend Snippet: Editing efficiencies of MAD7 and sgRNA constructs at the GUT1 locus.

Article Snippet: The gRNA scaffold sequences published by Inscripta were either found in the FAQ section of the Inscripta website, or in the sections about yeast and E. coli , respectively.

Techniques: Construct, CRISPR, Clone Assay, Disruption