base editing validation

[Gozde]

Hi everyone,

 

I am a beginner in genome editing.

I have been trying to optimize base editing in human primary dermal fibroblasts. Currently I am using plasmids;   47511 pFYF1320 EGFP Site#1 and 112099 pCMV_BE4max_P2A_GFP and following the protocol from Precision genome editing using cytosine and adenine base editors in mammalian cells. Nat Protoc 16, 1089–1128 (2021).

I've had a few trials with different gRNAs that I assumed failed (no difference in sanger sequencing trace files).

My question is do you think is it possible to validate genomic modification with sanger sequencing without enrichment ? 

My concern is to check that my gRNAs are working without enrichment by flow sorting and/or ngs analysis due to higher costs and availability. 

 

ps. I am using Neon transfection system and I optimized my transfection conditions with a GFP plasmid (pcDNA). 

3 days after transfection of gRNA plasmid and BE plasmid,  I isolated genomic DNA and tried to verify modification with sanger sequencing.  

 

I'd be glad if you'd have any comment and/or recommendation.

Thanks in advance for your support.

Gozde

[Karim Daliri]

Hi Gozde 

Try NGS , Sanger is not valid that much.

Bw

Karim

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[mengfang xia]

Hi Gozde

from my experience, it's fine to try sanger, However  enrichment is necessary, except that you have a high transfection efficiency.