Journal: Pathogens and Disease

Article Title: Nonspecific toxicities of Streptococcus pyogenes and Staphylococcus aureus dCas9 in Chlamydia trachomatis

doi: 10.1093/femspd/ftaa005

Figure Lengend Snippet: Structures of pTRL2(Δgfp) (A), pTRL2-Sp-dCas9 (B), pTRL2-Sp-dCas9 (C) and pTRL2-metG (D). Abbreviations: Sp, S. pyogenesis; Sa, S. aureus; dCas9, deactivated Cas9; MetG, methionine-tRNA ligase; Pgp, chlamydial plasmid-encoded protein; PdnaK, dnaK promoter; mKate, a monomeric basic far-red fluorescence protein; gRNA, guide RNA; PdefA, defA promoter; AmpR, penicillin/ampicillin-resistance gene; tetR, tet repressor; ColE1, E. coli replication origin E1; PtetA, tetracycline/anhydrotetracycline (ATC)-activated promoter. pL2 ori, CtL2 plasmid replication origin.

Article Snippet: The fixed cells were sequentially reacted with a mouse monoclonal anti-Sa-Cas9 primary antibody (Boster Biological Technology, catalog # {"type":"entrez-nucleotide","attrs":{"text":"M30929","term_id":"159088","term_text":"M30929"}} M30929 –3) and an FITC-conjugated anti-mouse IgG secondary antibody (Millipore Sigma, catalog # F9137).

Techniques: Plasmid Preparation, Fluorescence

Journal: FEMS Microbiology Letters

Article Title: Multiplex genome editing of microorganisms using CRISPR-Cas

doi: 10.1093/femsle/fnz086

Figure Lengend Snippet: (A) Cas9 and Cas12a expression and cleavage schemes. Left panel: Cas9 requires tracrRNA transcription and RNase III expression for CRISPR array transcript processing. Cas9 forms a complex with crRNA and tracrRNA and cleaves target DNA generating blunt ends. Right panel: Cas12a processes its own CRISPR array transcript to obtain individual crRNAs without the requirement of any tracrRNA or RNAse III co-expression. Cas12a stays in complex with crRNA and cleaves target DNA generating staggered ends. (B) Double strand break (DSB) repair mechanisms. DSBs can be repaired via non-homologous end joining (NHEJ), alternative non-homologous end joining repair pathways such as microhomology-mediated end joining (MMEJ), or via homologous direct recombination. NHEJ and MMEJ repair pathways can lead to the incorporation of deletions or insertions (only in case of NHEJ) in the targeted region. HDR is combined with the supplementation of donor DNA (dDNA), which can be double stranded or single stranded. dDNA can be used for insertion of long DNA sequences, deletion of genomic fragments, or introduction of single point mutations (SNPs).

Article Snippet: Fusarium fujikuroi [NJtech 02, CCTCC M2015614] , Sp Cas9 (constitutive),Plasmid expression (centromeric) , Several sgRNA expression cassettes (RNA pol III promoter, constitutive),Plasmid expression (centromeric) , – , Knockouts by disruption: 2, 20.8%; 3, 4.2% , (Shi et al . ) .

Techniques: Expressing, CRISPR, Non-Homologous End Joining

Journal: FEMS Microbiology Letters

Article Title: Multiplex genome editing of microorganisms using CRISPR-Cas

doi: 10.1093/femsle/fnz086

Figure Lengend Snippet: Multiplexed genome editing events in industrial microorganisms using CRISPR-Cas systems.

Article Snippet: Fusarium fujikuroi [NJtech 02, CCTCC M2015614] , Sp Cas9 (constitutive),Plasmid expression (centromeric) , Several sgRNA expression cassettes (RNA pol III promoter, constitutive),Plasmid expression (centromeric) , – , Knockouts by disruption: 2, 20.8%; 3, 4.2% , (Shi et al . ) .

Techniques: CRISPR, Expressing, Plasmid Preparation, Modification, Sequencing, In Vitro, Transformation Assay, Disruption

Journal: FEMS Microbiology Letters

Article Title: Multiplex genome editing of microorganisms using CRISPR-Cas

doi: 10.1093/femsle/fnz086

Figure Lengend Snippet: Multiplexing using gRNA polycistronic cassettes. (A) Expression of gRNAs from synthetic array dependent on Csy4 processing. In this case, Csy4 has to be co-expressed. (B) Expression of gRNAs from synthetic array dependent on endoribonuclease splicing. In most of the reviewed examples, these synthetic arrays are expressed using tRNAs as RNA pol III promoters. (C) Expression of gRNAs from native-like CRISPR array dependent on Cas9, tracrRNA and RNAse III processing. (D) Expression of gRNAs from native-like CRISPR array dependent on Cas12a processing.

Article Snippet: Fusarium fujikuroi [NJtech 02, CCTCC M2015614] , Sp Cas9 (constitutive),Plasmid expression (centromeric) , Several sgRNA expression cassettes (RNA pol III promoter, constitutive),Plasmid expression (centromeric) , – , Knockouts by disruption: 2, 20.8%; 3, 4.2% , (Shi et al . ) .

Techniques: Multiplexing, Expressing, CRISPR

Journal: FEMS Microbiology Letters

Article Title: Multiplex genome editing of microorganisms using CRISPR-Cas

doi: 10.1093/femsle/fnz086

Figure Lengend Snippet: Multiplexed genome regulation events in industrial microorganisms using CRISPRi and CRISPRa.

Article Snippet: Fusarium fujikuroi [NJtech 02, CCTCC M2015614] , Sp Cas9 (constitutive),Plasmid expression (centromeric) , Several sgRNA expression cassettes (RNA pol III promoter, constitutive),Plasmid expression (centromeric) , – , Knockouts by disruption: 2, 20.8%; 3, 4.2% , (Shi et al . ) .

Techniques: Expressing, Plasmid Preparation, Protein Binding, Activation Assay, Knock-Out, CRISPR

Journal: FEMS Microbiology Letters

Article Title: Multiplex genome editing of microorganisms using CRISPR-Cas

doi: 10.1093/femsle/fnz086

Figure Lengend Snippet: Characteristics of the most commonly used Cas orthologues for genome editing.

Article Snippet: Fusarium fujikuroi [NJtech 02, CCTCC M2015614] , Sp Cas9 (constitutive),Plasmid expression (centromeric) , Several sgRNA expression cassettes (RNA pol III promoter, constitutive),Plasmid expression (centromeric) , – , Knockouts by disruption: 2, 20.8%; 3, 4.2% , (Shi et al . ) .

Techniques: