Journal: Nature communications

Article Title: Insights into the compact CRISPR-Cas9d system.

doi: 10.1038/s41467-025-57455-9

Figure Lengend Snippet: Fig. 3 | The hybrid REM of Cas9d comprising the REC domains and Stem 2 and stem 3 of the sgRNA. a Structural alignment of the Cas9d (RNA-coordinated target Engagement Module, REM) with the SpyCas9 REC domain. b Close-up view of the Cas9d REM, as highlighted by the ellipse in a. c Interaction interface of REC domain

Article Snippet: The gene encoding the full-length Cas9d (Sangon) was codon optimized for E.coli expression and assembled into a modified pET vector (2Bc-T, Addgene #37236) with a C-terminal thrombin-TwinStrepII-Histag usingGibson assembly (NewEnglandBiolabs, E2611L).Mutations in Cas9d and SpyCas9 (WT expression plasmid, Addgene #101199) were introduced using the QuickChange Mutagenesis kit (Takara, Cat# 638949) following the manufacturer’s instructions.

Techniques: Drug discovery

Journal: Biotechnology and Bioengineering

Article Title: Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release

doi: 10.1002/bit.27200

Figure Lengend Snippet: Assessment of the diversity of Myr and PPYP flanking sequences and CRISPR‐derived mutations by DNA deep sequencing. (a) Schematic illustration of the pipeline established to identify CRISPR‐derived indel mutations in type‐C endogenous retrovirus (ERV) sequences from targeted DNA amplicon sequencing. Type‐C ERV specific primers were used to amplify approximately 300 bp surrounding the Myr or PPYP CRISPR target sites of the gag genes from untreated and CRISPR‐treated cells, and amplicons were analyzed by Illumina sequencing. Untreated reads were clustered as based on 97% sequence similarity to establish weighted profiles. Profiles were used to distinguish between natural ERV variations and indel mutations in CRISPR‐treated cells. (b, c) Clusters of Myr (panel B) or PPYP (panel C) deep sequencing reads of untreated parental CHO‐K1 cells. Clusters consisting of group 1, group 2 and group 3 type‐C ERV sequences are indicated in blue, purple and red lettering, respectively, according to the phylogenetic groups depicted in Figure . Clustered sequences expected to be targeted by CRISPR‐Cas9, as they contain the Myr2 sgRNA or PPYP6 sgRNA recognition sites and an adjacent PAM sequence, are shown in bold. The cluster representing the expressed group 1 type‐C ERV sequence is highlighted in yellow. (d) Number of distinct mutations and their corresponding read frequencies in seven clones (C02, D12, G09, A02, E10, K03, K14) isolated from Myr2 or PPYP6 sgRNA‐treated polyclonal populations, as indicated. Mutations of the expressed group 1 ERV, as previously detected in the mRNA in each clone, are indicated with a bold frame. Gray shaded boxes represent mutations occurring at a frequency higher than 0.4% (left‐hand side axis), thus implying the occurrence of the same mutation in more than one ERV locus, where the distinct ERV loci are separated by dotted lines. The estimated total number of mutated ERV loci of each clone is indicated by the right‐hand side axis. (e) Frequency of Myr2 or PPYP6 sgRNA‐induced repair junctions compatible with C‐NHEJ, alt‐EJ or HR DSB repair mechanisms. Repair junctions incompatible with these three main DSB repair mechanisms are grouped as Unknown. A total of 67 DNA repair junctions (n Myr = 45, n PPYP = 22) obtained from both Sanger cDNA and Illumina deep DNA sequencing were analyzed. (f, g) Proportion of the various mutations detected in each of the ERV sequence clusters shown in panels B and C, respectively. Clusters containing the Myr2 or PPYP6 sgRNA recognition sites including an adjacent PAM site are shown in bold letters as in panels B and C, while clusters with sgRNA possessing mismatches at position 13 or 15 in the sgRNA recognition site mismatches are shown in normal letters. The cluster representing the expressed group 1 type‐C ERV sequence is highlighted in yellow, as for panels B and C [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The mammalian codon‐optimized Streptococcus pyogenes Cas9 (SpCas9) nuclease expression plasmid JDS246 (Addgene plasmid #43861) was used to introduce site‐specific DSBs (Fu et al., ).

Techniques: CRISPR, Derivative Assay, Sequencing, Amplification, Illumina Sequencing, Clone Assay, Isolation, Mutagenesis, DNA Sequencing