CRISPR-CATCH is adapted to enrich for extrachromosomal DNA enabling phasing of genetic variants as well as ecDNA methylation profiling. NBThighlight
ecDNA isolation by CRISPR-CATCH
Genomic DNA was embedded in agarose plugs using a modified protocol based on guidelines from the manufacturer of the CHEF Mapper XA System as previously described. Briefly, molten 1% certified low-melt agarose in PBS was equilibrated to 45 °C. One million cells were pelleted per condition, washed twice with cold 1× PBS, resuspended in 30 µl PBS and briefly heated to 37 °C.
To perform in vitro Cas9 digestion, agarose plugs containing DNA were washed three times with 1× NEBuffer 3.1 with 5-min incubations. Next, DNA was digested in a reaction with 30 nM sgRNA and 30 nM spCas9 after pre-incubation of the reaction mix at room temperature for 10 min. To make two cuts on the native chromosomal locus, 15 nM of each sgRNA was added to the reaction.
To perform CRISPR-CATCH on flash-frozen patient tumor tissues, we removed frozen tissues from −80 °C and incubated them at −20 °C overnight. The tissues were thawed on ice, rinsed with MEM, Hanks’ Balanced Salts and cut into approximately 5 mm × 5 mm pieces using microdissection scissors. Molten 0.5% certified low-melt agarose in 1× PBS was equilibrated to 45 °C, and 50 µl was added to each plug mold .
For comparison between agarose-embedded DNA and in-solution HMW DNA, we performed HMW DNA extraction using the Qiagen MagAttract HMW DNA Kit following the manufacturer’s protocol. To digest linear DNA, we used Plasmid-Safe ATP-Dependent DNase and performed the reaction according to the manufacturer’s protocol over 5 days at 37 °C . After every 24 h, additional enzyme and ATP was added . After 5 days, DNase was inactivated by a 30-min incubation at 70 °C.
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