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CRISPR-Cas9 is frequently used for creating double-strand DNA breaks that result in indels through non-homologous end joining. Indels can revert to wild-type sequence and require sequencing or complex assays to measure. Cutting by two guide RNAs can lead to single indels at either cut site or simult …
- Michael Spagnuolo, Mark Blenner, Mark Blenner
- 2021
21 de ago. de 2021 · The mechanism of CRISPR/Cas-9 genome editing contains three steps, recognition, cleavage, and repair. The designed sgRNA recognizes the target sequence in the gene of interest through a complementary base pair.
- Misganaw Asmamaw, Belay Zawdie
- 2021
13 de out. de 2023 · Here, we present guidelines and recommended best practices for a step-by-step approach for the development and optimization of a genome editing pipeline for different crop species (Figure 1).
- 10.3390/plants12203564
- 2023/10
- Plants (Basel). 2023 Oct; 12(20): 3564.
- Design and Cloning of sgRNAs
- Transformation of Y. Lipolytica with CRISPR-Cas9 Plasmids
- Gene Excision Analysis
This protocol is used to design and clone sgRNAs that target two sites of interest for gene excision. sgRNAs are cloned into Cas9 episomal expression vectors optimized for Y. lipolytica. Variations of this method involving one plasmid with two sgRNA expression cassettes and two plasmids with a single sgRNA expression cassette on each have equal eff...
This protocol is used to transform CRISPR-Cas9 plasmids into Y. lipolytica. 1. 1. Prior to transformation, Y. lipolyticastored as a glycerol stock at −80 °C should be scraped and plated on a YPD agar plate. After 24 h of incubation at 28 °C, pick a single colony and inoculate 2 mL of liquid YPD media. Grow liquid cultures at 28 °C with shaking at 2...
This protocol is used to identify successful gene excision from Y. lipolytica clones and distinguish them from indel and non-mutated clones. A four-primer set is used as shown in Fig. 2to identify excised and non-excised genes in a single colony PCR reaction. This example focuses on determining gene excision of the PEX10 gene. The primers can be ea...
- Michael Spagnuolo, Mark Blenner, Mark Blenner
- 2021
10 de set. de 2020 · Generating excisions with paired CRISPR gRNAs is an attractive means to engineer complete loss of gene function, map regulatory regions, study 3D genome organization and model deletion-induced...
- Hannah L Watry, Carissa M Feliciano, Ketrin Gjoni, Gou Takahashi, Yuichiro Miyaoka, Bruce R Conklin,...
- 2020
20 de jan. de 2017 · The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system has emerged as the robust gene editing tool that functions through the double-stranded break repair process leading to targeted mutagenesis in higher genomes.
15 de jun. de 2021 · CRISPR immunity proceeds in three steps: adaptation, expression and interference. During adaptation, Cas proteins recognize potential spacer sequences (protospacers) in the bacteriophage genome and excise them in order to incorporate them within their own.
