CRISPR/Cas9 Gene Editing Services

CRISPR/Cas9 editing introduces a defined change at a defined position in the genome. That precision is what distinguishes it from the approaches that preceded it. Random plasmid integration places a transgene wherever it lands, producing clone-to-clone variation in expression from position effects and epigenetic silencing. RNA interference reduces expression but rarely eliminates it, and the residual protein is frequently enough to sustain the function under study.

Editing removes both limitations. A knockout eliminates the protein rather than reducing it, and a knock-in places a sequence at a chosen locus rather than at random. Altogen Labs provides CRISPR/Cas9 editing of mammalian cell lines, delivering characterized clonal lines rather than edited pools. To discuss an editing project, request a quote.

What can be made

Knockout lines eliminate expression of a target gene, and are the appropriate tool for establishing whether a phenotype depends on that gene rather than merely correlates with its expression. Knock-in lines introduce a defined sequence at a chosen locus, including point mutations that model a clinical variant, epitope tags for detection and purification, and reporter sequences placed under the control of an endogenous promoter rather than a constitutive one.

Targeted integration also provides a route to consistent transgene expression, since inserting at a defined locus avoids the clone-to-clone variability that random integration produces.

Design considerations

Guide RNA design determines both efficiency and specificity, and the two trade off against each other. Guides are selected against predicted off-target sites as well as on-target efficiency, with more than one guide typically designed per target so that a poorly performing guide does not stall the project.

For knock-in work, the repair template design governs the outcome. Homology arm length, the distance between the cut site and the intended edit, and silent mutations preventing re-cutting of the edited allele all affect the proportion of correctly edited clones and are settled before any transfection.

Zygosity is decided at the outset rather than discovered afterward. A heterozygous knockout in a gene where one allele suffices will show no phenotype, and a project that did not specify homozygosity as a requirement will frequently produce clones that cannot answer the question.

Clone isolation and validation

Editing a population produces a mixture of unedited, correctly edited, and variously mis-edited cells. Single cell cloning separates them, and validation establishes which clones carry the intended change.

Validation covers sequence confirmation at the target locus across all alleles, loss of protein by Western blot or capillary immunoassay for knockouts, and expression confirmation for knock-ins. Sequence confirmation alone is insufficient for a knockout, since an edit disrupting the reading frame at the target site does not always eliminate protein, and an alternative start site or splice variant can restore a truncated but functional product.

Off-target assessment at predicted sites is performed where the application requires it. Retaining more than one independently derived clone is recommended, since a phenotype present in one clone and absent in another is more likely a clonal artifact than a consequence of the edit.

Applied examples

Target validation. A knockout line is generated and compared with the parental line for sensitivity to a compound, establishing whether activity depends on the intended target rather than an off-target mechanism.

Clinical variant model. A point mutation observed in patients is introduced at the endogenous locus, producing a line expressing the variant under normal regulatory control rather than overexpressed from a plasmid.

Endogenous reporter. A reporter sequence is knocked in at a target locus so that expression reflects endogenous regulation, supporting pathway activity measurement in a physiologically relevant range.

Deliverables and documentation

Delivered lines are accompanied by sequence data across all alleles at the target locus, protein-level confirmation, passage history, and the guide and template sequences used. Lines are cryopreserved at low passage so subsequent work can return to characterized material, and cell banking is available where a line will support long-term work. Editing is performed on client-supplied or commercially obtained lines with appropriate permissions in place, and intellectual property considerations relating to the editing technology and the resulting lines are the responsibility of the client.

Send target gene, parental line, and intended edit, or request a quote.