Part:BBa_K5487104

yahK-sgRNA Box

What is yahK-sgRNA? sgRNA (single-guide RNA): An sgRNA is a synthetic RNA sequence designed to guide the Cas9 enzyme to a specific DNA sequence. It consists of two main parts: a 20-nucleotide sequence complementary to the target DNA (in this case, the yahK gene) and a scaffold sequence that binds to the Cas9 protein. Targeting the yahK Gene: yahK-sgRNA would be designed to specifically bind to a region within the yahK gene. When co-expressed with Cas9, the yahK-sgRNA directs the Cas9 nuclease to the yahK gene sequence, enabling targeted DNA cleavage. This cleavage can result in gene knockouts through non-homologous end joining (NHEJ) or gene modifications if a donor template is provided for homology-directed repair (HDR).

Applications of yahK-sgRNA: Gene Knockout Studies: By knocking out yahK, researchers can study the phenotypic effects and potentially identify the function of the yahK gene in bacterial cells. Genetic Engineering: Targeting yahK could be part of a larger strategy to genetically modify E. coli for synthetic biology applications, such as metabolic engineering or the development of microbial cell factories. Functional Genomics: It allows for the investigation of gene function in response to various environmental conditions or stresses.

How to Use yahK-sgRNA in Projects: Designing the sgRNA: Use bioinformatics tools to design the sgRNA sequence that specifically targets the yahK gene. Ensure minimal off-target effects by selecting unique sequences. Cloning into a CRISPR Vector: Insert the yahK-sgRNA sequence into a suitable CRISPR-Cas9 vector that expresses both the sgRNA and the Cas9 protein. Transformation and Expression in E. coli: Transform the CRISPR vector into E. coli strains and induce the expression of the yahK-sgRNA and Cas9. Selection and Screening: Use appropriate selection markers (e.g., antibiotic resistance) and screening methods (e.g., PCR, sequencing) to identify successful gene edits. Validation and Functional Analysis: Validate the gene knockout or modification by checking the absence or modification of yahK gene expression and perform phenotypic assays to study the impact of yahK disruption.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]