ANALYTICAL SERVICES FOR CRISPR/CAS9 EDITED CELL LINES

Analysis of CRISPR/Cas9 edited cells is done to check the efficacy of the system in introducing mutations (insertions, deletions, or substitutions) in the DNA sequences of the edited cell population.
SINGLE GUIDE RNA DESIGN AND PCR OPTIMIZATION

We design and optimize CRISPR/Cas9 single or double guide RNAs for genome editing projects in mammalian cell lines. The service can be requested together with gRNA validation or analytical services (see below for specification and prices) on CRISPR/Cas9 edited cell pools or clones.

ANALYTICAL SERVICES ON CRISPR/CAS9 EDITED CELL LINES

FINGEEC provides following analytical services on CRISPR/Cas9 edited cell pools or monoclonal cells:

  1. T7 nuclease (SURVEYOR) assay in polyclonal pool of edited cells
  2. Clonal sequence analysis with sanger sequencing
  3. TOPO cloning assay for clonal cell populations

 

1. T7 NUCLEASE MUTATION DETECTION ANALYSIS

Mutation detection analysis identifies and measures genome editing efficiency by detecting indels (insertions or deletions). Our services include:

  1. Genomic DNA extraction from KO cell pool: Genomic DNA from CRISPR edited cell KO pools are either extracted at FinGEEC (under Full service) or provided by the customer.
  2. Target Amplification: PCR amplification of edited genomic regions is done at FinGEEC. Heteroduplexes form when indels are present. Primers to amplify the target region can be designed and optimized by FinGEEC or are provided by customer (under Mutation detection only order).
  3. Indel Detection: Enzymes (e.g., T7 Endonuclease I) that recognize and digest non-perfectly matched DNA heteroduplexes, producing smaller DNA fragments.
  4. Fragment analysis: Using gel electrophoresis, we detect and quantify editing efficacy and predict genome editing outcomes.
2. CLONAL SEQUENCE ANALYSIS WITH SANGER SEQUENCING

Clonal sequence analysis with sanger sequencing is used to identify mutation (mismatches, insertions or deletions of nucleotides) within the single or double guide RNAs target region in single cell clones. The genomic DNA must be extracted from single cell clones after CRISPR/Cas9 editing in desired cells for a clean sequencing result. Our service includes:

  1. Genomic DNA extraction from single cell clone: Customer provides cell pellets or extracted genomic DNA from CRISPR edited single cell clones.
  2. Target Amplification: PCR is either optimized by customer or can be requested from us. Amplification of edited genomic region is done at FinGEEC. Sequencing primers to sequence the target region can be designed and optimized by FinGEEC or are provided by the customer.
  3. Sanger sequencing and result analysis: Using sanger sequencing, we analyze mismatches or indels and predict clonality of the genome editing.
PRICING INFORMATION
Validation services of genome editing (DNA)

Fee (€)

University of Helsinki

Fee (€)

Biocenter Finland

CRISPR guide RNA+PCR primer design and optimization 93 116
CRISPR guide RNA+PCR primer design and optimization (Extra order) 31 39
T7 nuclease mutation detection full service 202 251
T7 nuclease mutation detection full service (Extra order) 105 130
T7 nuclease mutation detection with PCR optimization (w/o gDNA isolation) 188 233
T7 nuclease mutation detection with PCR optimization (w/o gDNA isolation) (Extra order) 97 120
T7 nuclease mutation detection only (w/o gDNA isolation) 136 168
T7 nuclease mutation detection only (w/o gDNA isolation) (Extra order) 44 55
Clone sequence analysis with gDNA isolation 185 230
Clone sequence analysis with gDNA isolation (Extra order) 80 99
TOPO Clone analysis (single clone) 305 389
TOPO Clone analysis (single clone) (Extra order) 168 209
TOPO Clone analysis (polyclonal sample) 402 500
TOPO Clone analysis (polyclonal sample) (Extra order) 263 328
CITATION

Finnish Genome Editing Center (FinGEEC) should be mentioned in the acknowledgment in research publications containing data from CRISPR analytics performed by FinGEEC. Below are the templates of sentences that can be used: 

For analytical services including gRNA validation, T7 nuclease (SURVEYOR) assay, Sanger sequence analysis or TOPO assay:

"The xx analysis of xx (genes/KO/KIs) was performed by Finnish Genome Editing Center (FinGEEC), Research Programs Unit, Faculty of Medicine, University of Helsinki, supported by HiLIFE, University of Helsinki and Biocenter Finland."