Custom CRISPR Knockout and Knock-In Cells
Using CRISPR technology, Cellecta offers services to make cell lines with targeted gene knockouts or containing specific knock-in sequences. The approach for each project can vary depending on what cell engineering is required. However, our general knockout and knock-in services are outlined below.
Knockout Cell Lines
Cellecta offers custom developed knockout cell lines using CRISPR technology. With this service, we will make a few sgRNA knockout constructs targeting your gene of interest and introduce these into the parental cells to generate cell pools few pools. After checking each putative knockout cell pool by sequencing to see which sgRNA produce rearrangements at the target site. We then isolate/expand monoclonal cell lines, assay these by sequencing and provide you knockout clones with out-of-frame indels in all alleles.
Our standard knockout protocol uses a Zero-Footprint CRISPR-based approach where both the sgRNA and Cas9 expressing elements are introduced transiently into the cells to generate the knockout. Since they do not integrate into the genomic DNA, no foreign genetic material is introduced into the genome. Only the desired indels in the target gene of interest are permanent introduced.
Of course, if you do want stable integration and expression of either the sgRNA or Cas9 instead, we can accommodate this using standard lentiviral constructs. For example, for some assays, it may be sufficient to use a cell pool where the target gene is knocked out in a significant portion of the cells. For this purpose, it may be preferable to use integrated lentiviral constructs with the sgRNA and Cas9 elements to make it possible to select for cells containing these elements during expansion.
Also, for some studies inducible knockout cell lines may be preferable. In this case, we can engineer cells that express the Cas9 protein and have an tetracycline inducible sgRNA. These can “turned on” at a certain point in the experiment to induce disruption of the target gene. This will not produce 100% knockout across the whole cell population but may be useful for some studies.
Knock-In Cell Lines
Like knockout cell lines, knock-in modifications require CRISPR sgRNA construct. In addition, however, a donor construct is required to insert the DNA sequence is interest into the target site. With this service, we design and construct an appropriate donor for the desired modification, and introduce it, with the guide DNA, into your parental cells of choice. The resulting pools are tested for rearrangement at the target site and then selected individual cells are isolated and expanded to identify cell clones with the specific modifications.
Flexibility and Quality
With knockout or knock-in projects we offer flexible services depending on your needs and budget. We can provide you cell pools with confirmed rearrangements which you can use directly for certain assays or to perform your own clonal isolation, selection, and confirmation to make a monoclonal line.
Regardless of project, we provide validated cell pools or clonal cell lines. For the cell pools, we sequence and confirm modifications at the target location. For clonal lines, we characterize the specific indels or insertions present in each allele and, for knockout cells in particular, check for the presence of the wild-type allele since many common cell lines are polyploid.
To discuss a specific project, please contact us.