iCyagen AI-Gene Editing Designer User Guide

iCyagen AI-Gene Editing Designer is a multi-functional intelligent agent designed for cell and animal experimental scenarios. It automates the generation of standardized gene editing strategies, including knockout (KO), point mutations, and cell line knockouts in rodent models. Covering basic research, mechanism analysis, and functional validation, it aims to lower the barrier to gene editing design and provide researchers with efficient, precise, and actionable outputs.

We support knockout design for mouse and rat strains, covering three main modes: Constitutional Knockout (KO), Conditional Knockout (cKO), and ES-cKO. These strategies can be tailored to various research requirements.

• Type 1: Constitutional Knockout (KO)
  Targeting the core exons of the gene in C57BL/6JCya mice or SD rats, this strategy uses frameshift mutations to trigger Nonsense-Mediated mRNA Decay (NMD), resulting in a permanent, whole-body loss of gene function.
• Type 2: Conditional Knockout (cKO)
  Utilizing the Cre-loxP site-specific recombinase system in C57BL/6JCya mice or SD rats, this approach enables tissue-specific or time-specific gene deletion. It is the preferred model for studying refined gene functions and bypassing embryonic lethality caused by whole-body knockout.
• Type 3: ES Cell-based Conditional Knockout (ES-cKO)
  Specifically for C57BL/6JCya mice, this strategy leverages Embryonic Stem (ES) cell targeting combined with conditional knockout logic. It offers high targeting precision, low off-target risk, and controllable chimerism.

• Required Parameters: Species, Gene Name or NCBI Gene ID, Strategy Type.
• Prompt Example: "Design an ES-cKO strategy for the mouse Trp53 gene."

Specifically designed for microRNA research, this strategy adapts to microRNA regulatory mechanisms and is applicable to disease development studies. It currently supports C57BL/6JCya and C57BL/6NCya mouse strains.

• Required Parameters: microRNA Name, Mouse Strain.
• Prompt Example: "Construct a knockout strategy for mir145 in C57BL/6NCya mice."

We support point mutation design for mouse, rat, and human genes. Mutation types include single nucleotide substitutions, insertions, or deletions. Applications range from disease modeling and pathogenic variant validation to functional site mapping.

• Mouse: C57BL/6JCya, C57BL/6NCya
• Rat: SD, F344, Wistar, and other mainstream strains.
• Humanized Mouse Models: C57BL/6JCya, C57BL/6NCya.

Single base substitution, small fragment insertion, small fragment deletion, and complex indel (insertion + deletion) mutations.

• Required Parameters: Species, Strain, Gene Name/ID, Specific Mutation Site details.
• Prompt Example: "Construct a point mutation strategy for Trp53 in C57BL/6NCya mice; I will provide the specific mutation site details in a follow-up."

This module designs knockout strategies for specific cell lines (mouse, rat, or human). It ensures a rigorous match between species and cellular background to output standardized, actionable experimental protocols for in vitro functional validation, mechanism exploration, and drug target screening.

• Required Parameters: Species, Gene Name/ID, Target Cell Line Name.
• Prompt Example: "Design a gene knockout strategy for Trp53 in the SH-34 cell line."

Definition: Constitutional (Whole-body) Knockout refers to the permanent deletion of a target gene across all cells of the organism using gene editing technology.

• The target gene is absent in all tissues.
• Ideal for studying the global role of a gene in development and physiology.

• May result in embryonic lethality or severe developmental defects, preventing the generation of viable adult animals.
• Cannot differentiate gene function across different tissues or developmental stages.

Global functional assessment; study of non-lethal genes.

Definition: Conditional Knockout (cKO) uses spatiotemporal control to delete a gene in specific tissues or at specific time points, bypassing the lethal or pleiotropic effects of a global knockout.

Two loxP sites are inserted in the same orientation flanking critical exons of the target gene (a "floxed" gene). Under normal conditions, floxed mice exhibit wild-type phenotype and physiology.

By crossing a floxed mouse with a Cre-recombinase expressing mouse (e.g., tissue-specific Cre or inducible CreERT2), the Cre protein recognizes the loxP sites and "cuts out" the intervening exon, achieving targeted gene deletion.

• Avoids embryonic lethality.
• Enables precise study of gene function in specific cell types or developmental phases.
• Flexible control over the "where" and "when" of gene deletion.

• Tandem Repeats:
  DNA sequences where two or more base pairs are repeated and the repetitions are adjacent to each other. They play key roles in genome stability, regulation, and genetic diversity.
• NMD (Nonsense-Mediated mRNA Decay):
  A cellular surveillance mechanism that detects and degrades mRNA transcripts containing premature stop codons (PTCs), preventing the translation of truncated and potentially toxic proteins.
• Wild-type Littermates:
  Animals born in the same litter as the gene-edited offspring but without the target mutation. They serve as ideal controls to minimize environmental and genetic background variables.
• EM (Embryonic Mutation):
  Mutations introduced during early embryonic development. These are typically present in all cells of the embryo, leading to constitutional knockout or mutation.
• Dot Plot (Sequence Analysis):
  A visual method for comparing two sequences to identify regions of similarity. In genomics, it helps identify repeats, insertions, deletions, or inversions.
• GC Content Analysis:
  Calculation of the percentage of Guanine (G) and Cytosine (C) bases within a DNA/RNA sequence. High or low GC content affects sequence stability and the efficiency of experimental designs like PCR or CRISPR targeting.