CRISPR/Cas projects initiated with the core will be screened for potential off-target genome editing events in N1 animals using Sanger sequencing of PCR amplicons flanking predicted off-target sites. The core will design PCR primers to amplify a specified number of sites based on consultation with the investigator. Typically, the core recommends to screen 2-4 N1 mice at 5-10 off-target sites, however, there is no set number of mice or sites for each project. The core will analyze the sequencing traces and redesign the PCR scheme if the results are ambiguous.
What Will Happen
- The investigator will initiate a project in iLab for Targeted Analysis of Off-Target Mutagenesis. The investigator will specify the number of animals they have for screening and the number of sites they wish to be screened. An account number must be provided at this time.
- The investigator will collect tissue from the mice to be screened and arrange with the core to drop off the tissue to ABBR R851A. Alternatively, the investigator can supply the core with extracted DNA samples. The core requests an additional WT control sample for investigator extracted DNA. The control sample will not be charged for analysis.
- The core will have oligonucleotides for PCR synthesized by an approved vendor.
- If necessary, the core will extract DNA from the supplied tissues and resuspend the DNA in nuclease-free water. After screening is completed, the investigator can arrange with the core to have aliquots of the DNA for their own genotyping, if desired.
- The core will perform standard PCR on the extracted DNA samples for the designed off-target sites.
- The core will clean up the PCR reactions with the anticipated product size for the desired allele and send out samples for Sanger sequencing.
- The core will analyze the Sanger sequencing results to confirm the absence heterozygous sequence. The presence of heterozygous sequence would indicate a genome editing event.
- After analyzing the samples at all of the specified off-target sites, the core will provide the investigator with a report, including the genome locations of all off-target sites screened, the number of mismatches for each predicted site, the sequencing primers, and the results for each mouse analyzed by predicted site.
What to Expect
- The core will provide off-target analysis results from predicted off-target sites primarily based on information available from the Wellcome Sanger Institute Genome Editing (WGE) Website (https://wge.stemcell.sanger.ac.uk/). This website predicts off-target sites with base mismatches to the target sequence and uses a canonical PAM (NGG). If the investigator wishes to screen off-target sites based on other prediction algorithms from guide RNA selection websites, they can provide the genomic coordinates for each predicted site to be screened.
- If the PCR reactions fail to produce products with appropriate controls, new primers will be designed and PCR repeated before any sequencing is attempted.
- Wild-type controls will also be sequenced to ensure that the Sanger sequencing results are the same as the predicted sequence from available genome browsers.
- If any sample fails to produce a quality trace by Sanger sequencing, repeat Sanger sequencing attempts will be submitted until the core can accurately assess the sequence for the sample at the specific locus.