Journal: Cell reports

Article Title: Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome

doi: 10.1016/j.celrep.2021.109930

Figure Lengend Snippet: (A)GFP+ E. coli exhibit a sick colony morphologyafter infection with M13 phage carrying GFP-targeting (GFPT) CRISPR-Cas9. NT (non-targeting) or GFPT M13 were used to infect Sm R W1655 F+ sfgfp or Sm R W1655 F+ mcherry as a control. Cells were infected, diluted, and spotted onto media with selection for the vector; f1A or f1B indicates vector version. (B) CRISPR-Cas9 targeting the sfgfp gene can induce loss of fluorescence. Colonies arising from infection with NT-M13 or GFPT-M13 were subjected to several rounds of streak purification on selective media to ensure phenotypic homogeneity and clonality. The majority (11/16) of GFPT clones exhibited a loss of fluorescence. (C) Clones exhibiting loss of fluorescence either lack an sfgfp PCR amplicon or exhibit an amplicon of decreased size. Genomic DNA was isolated from streak-purified clones, and PCR was used to determine whether the sfgfp gene was present. PCR for the 16S rRNA gene was performed as a positive control. (D) Genome-sequencing results confirm that non-fluorescent clones have chromosomal deletions encompassing the targeted gene. Read depth surrounding sfgfp locus for a fluorescent control clone G9 (green line) and all non-fluorescent clones (gray lines). Deletion size is indicated in red; range indicates a deletion flanked by repetitive sequences. Black arrow and vertical line denote position of targeting. See also .

Article Snippet: Plasmid: pCas9 (Low-copy vector carrying cas9 , tracrRNA, and CRISPR array; Cm R ) , , RRID: Addgene_42876.

Techniques: Infection, CRISPR, Selection, Plasmid Preparation, Fluorescence, Purification, Clone Assay, Amplification, Isolation, Positive Control, Sequencing

Journal: Cell reports

Article Title: Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome

doi: 10.1016/j.celrep.2021.109930

Figure Lengend Snippet: (A) M13-delivered GFPT CRISPR-Cas9 leads to reduced competitive fitness of the GFP-marked strain. A co-culture of Sm R F+ sfgfp and Sm R F+ mcherry was incubated with NT-M13 or GFPT-M13 at a starting MOI of ~500. Carb was added to a final concentration of 100 μg/mL to select for phage infection. Co-cultures were sampled every 4 h over 24 h; cells were washed, serially diluted, and spotted onto non-selective media to assess targeting of the GFP-marked strain. (B) Carb in culture supernatants was not detectable within 4 h of growth using a Carb bioassay against indicator strain Bacillus subtilis 168; bioassay detection limit approximately 2.5 μg/mL. (C) Flow cytometry of co-cultures 8 h following the addition of phage and Carb show reduced GFP+ events in the GFPT versus NT condition. Representative flow plots show data from 1 of 3 biological replicates. Inset: bar graph quantifying percentage of GFP+ and mCherry+ events for 3 replicates (left); plating results for a single replicate on non-selective media (right). (D) GFPT CRISPR-Cas9 changes the shape of the distribution of GFP+ population. Histogram of mCherry+ and GFP+ events by intensity shows that a proportion of GFP+ cells in the GFPT condition have shifted to a state of lower fluorescence. Bars indicate the mean of 3 biological replicates; connected points are individual replicates. See also .

Article Snippet: Plasmid: pCas9 (Low-copy vector carrying cas9 , tracrRNA, and CRISPR array; Cm R ) , , RRID: Addgene_42876.

Techniques: CRISPR, Co-Culture Assay, Incubation, Concentration Assay, Infection, Flow Cytometry, Fluorescence

Journal: Cell reports

Article Title: Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome

doi: 10.1016/j.celrep.2021.109930

Figure Lengend Snippet: (A) Sanger-sequencing results confirm the expected spacer present in phagemid DNA extracted from fluorescent yellow isolates (Y1) colonizing NT mice (M1, M4, M5, M6, M7, M8, and M10) and fluorescent red isolates (R1 and R2) colonizing GFPT mice (M13, M14, and M18). In contrast, 4 out of 5 fluorescent yellow isolates colonizing GFPT mice (M11, M13, M14, M16, and M18) were confirmed to have lost the spacer. No Sanger-sequence data were obtained for the last isolate (M13) with the report for failing being “no priming,” suggesting loss of a larger fragment from the phagemid. (B) Diagnostic digest of CRISPR-Cas9 phagemid DNA indicates loss of a portion of phagemid DNA for the phagemid extracted from M13 Y1. Expected fragment sizes from KpnI-XbaI double digest: 5,289, 3,285, and 2,573 bp. (C) Genome-sequencing data for M13 Y1 confirms loss of DNA from phagemid. Sequencing coverage across the GFPT phagemid reveals lack of reads corresponding to the cas9 gene and parts of the CRISPR array and tracrRNA.

Article Snippet: Plasmid: pCas9 (Low-copy vector carrying cas9 , tracrRNA, and CRISPR array; Cm R ) , , RRID: Addgene_42876.

Techniques: Sequencing, Diagnostic Assay, CRISPR

Journal: Cell reports

Article Title: Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome

doi: 10.1016/j.celrep.2021.109930

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmid: pCas9 (Low-copy vector carrying cas9 , tracrRNA, and CRISPR array; Cm R ) , , RRID: Addgene_42876.

Techniques: Clone Assay, Recombinant, Ligation, Sequencing, CRISPR, Plasmid Preparation, Expressing, Software

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