Frequently, homozygous and heterozygous mutations have different biological effects, and it can be useful to isolate both types of mutants in a gene editing project for disease modeling. Using the CRISPR-SNIPER technology, both heterozygous and homozygous mutants can easily be isolated from in one experiment.
Purpose: Produce a knockout mutation Cells: Human iPS cells Knock-in donor: Δ56 bp deletion donor
Above: Generation of wild type, homozygous, and heterozygous mutants in a single CRISPR-SNIPER experiment.
Selective mutation of similar genes
Similar genes with high homology can be difficult to selectively mutate. Adding Nickase can increase the stringency of the guide, at a cost of reduced overall efficiency. Using the SNIPER method, we can increase the overall mutation rate and readily isolate mutant clones.
Purpose: To distinguish HLA-A and HLA-B mutants Cells: HCT-116 Analysis: qPCR analysis of bulk genomic DNA
Above: Generation of mutations using a HLA-A specific guide RNA (left panel) and a guide RNA common to both HLA-A and HLA-B (right panel). The specific guide RNA results in selective knock-down of HLA-A, while the common guide RNA knocks down both HLA-A and HLA-B.
Editing multiple genes in one experiment
Creating multiple mutations in one cell usually requires multiple rounds of gene editing. For cells that do not do well with extended passaging, such as iPS cells, multiple passaging can lead to slow growth, formation of genetic abnormalities, and difficulty in differentiation.
We can generate multiple mutations in one experiment by incorporating multiple gRNAs in one optimized experiment using SNIPER to isolate the rare desired mutations from the pool.
Purpose: Creation of five knock-out mutations in a single experiment Cells: HCT-116 Analysis: qPCR analysis of bulk genomic DNA
Above: gRNA for five different genes were included in one optimized transfection, resulting in the creation of knock-out mutations in five genes in one experiment. This minimizes the passage number.
Using long donor DNA for difficult case
Difficult cases with low mutation frequency, such as knock-ins with long insertion sequences, can be difficult to handle with traditional CRISPR methods. Using long donor DNAs can greatly increase mutation frequency due to increased efficiency of homologous recombination. However, long donors are expensive, difficult to synthesize, prone to generation of non-specific mutants, and difficult to analyze using traditional qPCR probes.
Incorporation the SNIPER technology allows for improved screening for correct mutants, and SNIPER probes hybridize better to long donors, resulting in greatly increased mutation frequency.
Results of a comparison study
ca. 50 bp
ca. 1000 bp
Insertion of large gene fragments
Creating knock-ins with large insertions (>2 kbp) the mutation rate can drop significantly. We can optimize the editing conditions using SNIPER to optimize the mutation conditions. SNIPER allows the creation of mutants with insertions of 5-7 kbp or larger.
One situation where this has proved useful is in the creation of GFP reporters in stem cells. Rapid optimization of differentiation conditions can be achieved using GFP inserted into differentiation markers. GFP-expressing stem cells can also be used for tracking specific cell populations during the differentiation process.
Generation of iPSC-based disease models.
Genome editing is ideal for the creation of iPSC-based disease models for genetic diseases. Using CRISPR-SNIPER to introduce a disease causing mutation into iPSCs from healthy donors allows researchers to isolate the effects of the mutation. Conversely, iPSCs from patients can be edited to revert a mutant gene to the healthy genotype. Either case has a built in isogenic control (the healthy iPSCs) to facilitate study of the effects of the mutation on disease function and to provide a control line for cell-based screening using disease model cells to identify new pharmaceutical agents.
Generation of iPSC-based disease models using CRISPR-SNIPER