Hi everyone!

I am working with a genome-wide guide RNA (gRNA) pooled library (Tzelepis et al. 2016) with 90.000gRNAs targeting all the genome to perform drug screens in human cells (Addgene library: https://www.addgene.org/pooled-library/yusa-crispr-knockout-human-v1/).

Once I got the CRISPR mutant gRNA library setup, I added the drug and waited for single resistant colonies to appear to pick them and analyze them individually.

MY PROBLEM is when looking for inserted gRNAs in each resistant colony I keep picking (50% of the times) an unknown sequence which is longer than an usual gRNA (34bp instead of18-20bp). I normally amplify the resistant colony DNA by PCR (the region of the vector in which the gRNAs are inserted), subclone the PCR product in single vectors and Sanger-sequence. The strange sequence (pic attached) is not in the original database of the gRNAs library and when doing a BLAST we are not able to find it either. Sequence is: '-CGCACCGTGGGCTTGTACTGTTTTAGAGCTAGAA-'

Is anyone having the same problem or know if this problem is caused by me when cloning or amplifying the DNA library?

THANKS IN ADVANCE :D

Ana Monfort-Vengut