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Series GSE271068 Query DataSets for GSE271068
Status Public on Oct 16, 2024
Title AAV vector-derived elements integrate into Cas9-generated double strand breaks and disrupt gene transcription
Organism Mus musculus
Experiment type Other
Summary We previously found that an adeno-associated virus (AAV) vector containing S. aureus Cas9 and a multi-target guide (g)RNA could integrate into the genome and prematurely terminate transcription of Ube3a-ATS, a long non-coding RNA. Here, we assessed the performance of three additional AAV vectors containing S. aureus Cas9 and twenty-five vectors containing N. meningococcus Cas9, all targeting single sites within Ube3a-ATS. We found that none of these single-target gRNA vectors were as effective as multi-target gRNA vectors at reducing Ube3a-ATS expression in neurons. We also developed a new anchored multiplex PCR sequencing (AMP-seq) method and analysis pipeline to quantify the relative frequency of all possible editing events at target sites, including unresolved double-stranded breaks (DSBs) and capture of foreign DNA. We found that integration of AAV was the most frequent editing event (67-89% of all edits) at three different single target sites, far surpassing insertions and deletions (indels). None of the most frequently observed indels was capable of blocking transcription when incorporated into a Ube3a-ATS minigene reporter, whereas two vector derived elements—the polyA and reverse promoter—reduced downstream transcription by up to 50%. Since not all editing events disrupt gene transcription, our findings suggest that the probability that a gene trapping AAV integration event occurs is influenced by which vector-derived element(s) are integrated and by the number of target sites.
 
Overall design AMP-seq on DNA from neurons treated with various NmCas9-gRNA AAVs: NmCas9-AAV gRNA23 (0-7 day treatment), NmCas9-AAV gRNA25 (7 day treatment), NmCas9-AAV gRNA21 (7 day treatment), NmCas9-AAV non-targeting gRNA (7 day treatment), or untreated DNA. AMP-seq on DNA from neurons of P1 (post-natal-day 1) C57BL/6 mice treated with SaCas9-gRNA AAVs (SaCas9-AAV-G3) or SaCas9-AAV non-targeting gRNA.
 
Contributor(s) Bazick HO, Mao H, Niehaus JK, Wolter JM, Zylka MJ
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Submission date Jun 28, 2024
Last update date Oct 16, 2024
Contact name Austin J Hepperla
E-mail(s) hepperla@unc.edu
Organization name University of North Carolina at Chapel Hill
Department Genetics
Street address 7018B Mary Ellen Jones Building
City Chapel Hill
State/province NC
ZIP/Postal code 27599
Country USA
 
Platforms (1)
GPL23969 Illumina MiniSeq (Mus musculus)
Samples (60)
GSM8368070 NmCas9-AAV gRNA 21, 7 Day Treatment; Rep1
GSM8368071 NmCas9-AAV gRNA 21, 7 Day Treatment; Rep2
GSM8368072 NmCas9-AAV gRNA 21, 7 Day Treatment; Rep3
Relations
BioProject PRJNA1129539

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE271068_Bazick_AMP-seq_read_counts.xlsx 13.0 Kb (ftp)(http) XLSX
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