Journal: Frontiers in Genetics

Article Title: FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella

doi: 10.3389/fgene.2021.738746

Figure Lengend Snippet: The diagram for genetic manipulation of FnCas12a/crRNA-mediated genome editing in E. tenella . (1) The generation of plasmids used as homologous recombination template. (2) Expression and purification of FnCas12a proteins. (3) crRNA synthesis in vitro . (4) The purification of E. tenella sporozoites. (5) E. tenella sporozoites were transfected with FnCas12a protein, crRNA with or without homologous recombination template using Lonza 4D-Nucleofector. (6) Sporozoites (2×10 6 ) were cultured in PCK cells to show transfection results. We also used this step to compare the transfection efficiency of different nucleofector programs. (7) The remaining part of sporozoites (8×10 6 ) was inoculated in chicken via cloaca and selected with pyrimethamine in feed. The progeny oocysts were harvested from feces 6–10days after inoculation. (8) The progeny oocysts infected new batches of chickens (coccidia-free) by oral gavage. Pyrimethamine was used to enrich for parasites carrying the DHFR gene.

Article Snippet: The FnCas12a protein was purified using the BeyoGoldTM His-tag Purification Resin (P2210, Beyotime Biotechnology) according to the manufacturer’s instructions.

Techniques: Homologous Recombination, Expressing, Purification, In Vitro, Transfection, Cell Culture, Infection

Journal: Frontiers in Genetics

Article Title: FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella

doi: 10.3389/fgene.2021.738746

Figure Lengend Snippet: FnCas12a-mediated gene knock-out in E. tenella . (A) The effect of the molar ratio of RNP/template on in vitro cleavage efficiency of the EtHistone H4 fragment. The molar ratio of RNP/template at 0:1, 2:1, 4:1, 10:1, 20:1, 30:1, 40:1, respectively, with the temperature at 37°C and reaction time of 60min. (B) The effects of incubation times. The reaction time of 10, 20, 30, 60, 90, and 120min with the temperature at 37°C and the molar ratio of RNP/template at 30:1. (C) The effects of reaction temperature for the cleavage. The reaction at the temperature of 16, 28, 37, 41, 44, 50, 60, 70, and 80°C, respectively, with the molar ratio of RNP/template was 30:1 and reaction time of 10min. (D) Comparison of cleavage activity of three crRNAs. The molar ratio of RNP/template at 10:1, incubated in 37°C for 60min. The edited products were quantified with agarose gel electrophoresis. (E) Schematic diagram of FnCas12a/crRNA-1 trigger genome editing in EtHistone H4 . (F) The top 10 indels were obtained via next-generation sequencing.

Article Snippet: The FnCas12a protein was purified using the BeyoGoldTM His-tag Purification Resin (P2210, Beyotime Biotechnology) according to the manufacturer’s instructions.

Techniques: Knock-Out, In Vitro, Incubation, Comparison, Activity Assay, Agarose Gel Electrophoresis, Next-Generation Sequencing

Journal: Frontiers in Genetics

Article Title: FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella

doi: 10.3389/fgene.2021.738746

Figure Lengend Snippet: FnCas12a-mediated EtActin gene knock-in. (A) Schematic diagram illustrates FnCas12a-mediated knock-in at EtActin . E. tenella sporozoites were transfected with FnCas12a-RNP and homologous recombination template (P2A-DHFR-EYFP cassette in-frame with C-terminal of EtActin gene). (B) Schematic diagram of FnCas12a crRNA-DNA-targeting complex. The target sequence is in blue, and the PAM sequence is in pink. (C) In vitro cleavage of PCR products harboring the targeting sequence at EtActin . (D) PCR confirmation of EtActin tagged E. tenella and parental one (wild-type, WT). The positions of primers (F1/R1; F2/R2; and F3/R3) are shown in (A) . (E) Sporozoites of EtActin knock-in were cultured in MDBK cells and EYFP expression at different time points (24, 48, and 72h) after sporozoites invasion. (F) The cecal smears of EtActin knock-in were prepared in 96, 120, 144, and 168h (h.p.i.) for detection of merozoites, unsporulated oocyst, respectively. UO, unsporulated oocyst; SO, sporulated oocyst.

Article Snippet: The FnCas12a protein was purified using the BeyoGoldTM His-tag Purification Resin (P2210, Beyotime Biotechnology) according to the manufacturer’s instructions.

Techniques: Gene Knock-In, Knock-In, Transfection, Homologous Recombination, Sequencing, In Vitro, Cell Culture, Expressing

Journal: Cell reports

Article Title: Harnessing noncanonical crRNAs to improve functionality of Cas12a orthologs

doi: 10.1016/j.celrep.2024.113777

Figure Lengend Snippet:

Article Snippet: pUC19-U6-FnCas12a crRNA-BsmBI cassette (BPK4446) , Kleinstiver et al. , Addgene plasmid #114087.

Techniques: Virus, Recombinant, Protease Inhibitor, DNA Extraction, Mutagenesis, Nucleic Acid Purification, Cloning, Plasmid Preparation, Software