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Series GSE127752 Query DataSets for GSE127752
Status Public on Mar 08, 2019
Title Multiplexed Cas9 targeting reveals genomic location effects and gRNA-based staggered breaks influencing mutation efficiency and patterns
Organism Mus musculus
Experiment type Expression profiling by high throughput sequencing
Third-party reanalysis
Summary Understanding the impact of guide RNA (gRNA) and genomic locus on CRISPR/Cas9 activity is crucial to design effective gene editing assays. However, it is challenging to profile Cas9 activity in the endogenous cellular environment. We leverage our TRIP technology to integrate ~1k barcoded reporter genes in the genomes of mouse embryonic stem cells. We target the integrated reporters (IRs) using RNA-guided Cas9 and characterize induced mutations by sequencing. We report that gRNA-sequence and IR locus explain most variation in mutation efficiency. Predominant insertions of a gRNA-specific nucleotide are consistent with template-dependent repair of staggered DNA ends with 1-bp 5′ overhangs. We confirm that such staggered ends are induced by Cas9 in mouse pre-B cells. To explain observed insertions, we propose a model generating primarily blunt and occasionally staggered DNA ends. Mutation patterns indicate that gRNA-sequence controls the fraction of staggered ends, which could be used to optimize Cas9-based insertion efficiency.
Overall design Mutation profiling upon RNA-guided Cas9 targeting of (i) mouse embryonic stem (mES) cell line, EStTA-PB-B18, containing the same 36 mPGK-driven TRIP barcoded reporter integrations per cell (EStTA-PB-B18); (ii) TRIP cell pool containing ~1k barcoded reporters integrated heterogeneously throughout the genomes of the cells in the pool (GEMP-O). Cell line was targeted in independent experiments using three single-guide RNAs (sgRNA1-3). Pool was targeted in independent experiments using sgRNA2 with or without a custom-designed ssODN, and with sgRNA3. We also did expression profiling of the TRIP cell line and pool before Cas9 targeting (see GEO accession GSE48606 for cell line expression, and this submission for the pool expression). Files related to re-analyzed samples from GSE48606 are linked below as supplementary files.
Contributor(s) Gisler S, Goncalves JP, Akhtar W, de Jong J, Pindyurin AV, Wessels LF, van Lohuizen M
Citation(s) 30962441
Submission date Mar 03, 2019
Last update date Apr 17, 2019
Contact name Joana P Goncalves
Organization name Delft University of Technology
Department Intelligent Systems
Street address Van Mourik Broekmanweg 6
City Delft
ZIP/Postal code 2628 XE
Country Netherlands
Platforms (2)
GPL16417 Illumina MiSeq (Mus musculus)
GPL17021 Illumina HiSeq 2500 (Mus musculus)
Samples (49)
GSM3638082 Multi-promoter TRIP pool normalization reads, repl 1, PCR 1
GSM3638083 Multi-promoter TRIP pool normalization reads, repl 1, PCR 2
GSM3638084 Multi-promoter TRIP pool normalization reads, repl 2, PCR 1
Reanalysis of GSM1207441
Reanalysis of GSM1207452
Reanalysis of GSM1207463
Reanalysis of GSM1207474
BioProject PRJNA525279
SRA SRP187354

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
GSE127752_crisprTRIP_EStTA-PB-B-18_2605_sgRNA3_indels.txt.gz 2.6 Mb (ftp)(http) TXT
GSE127752_crisprTRIP_EStTA-PB-B-18_2794_sgRNA1_indels.txt.gz 3.1 Mb (ftp)(http) TXT
GSE127752_crisprTRIP_EStTA-PB-B-18_2794_sgRNA2_indels.txt.gz 3.0 Mb (ftp)(http) TXT
GSE127752_crisprTRIP_multiTRIP-GEMP-O_3297_sgRNA3_indels.txt.gz 261.5 Kb (ftp)(http) TXT
GSE127752_crisprTRIP_multiTRIP-GEMP-O_3593_2_sgRNA2+ssODN_indels.txt.gz 189.9 Kb (ftp)(http) TXT
GSE127752_crisprTRIP_multiTRIP-GEMP-O_3593_5_sgRNA2_indels.txt.gz 181.4 Kb (ftp)(http) TXT
GSE127752_crisprTRIP_multiTRIP-GEMP-O_additionalHR1_3284_sgRNA2+ssODN_indels.txt.gz 238.8 Kb (ftp)(http) TXT
GSE127752_crisprTRIP_multiTRIP-GEMP-O_additionalHR2_3593_1_sgRNA2+ssODN_indels.txt.gz 211.0 Kb (ftp)(http) TXT
GSE127752_crisprTRIP_multiTRIP-GEMP-O_additionalHR3_3593_3_sgRNA2+ssODN_8uM_indels.txt.gz 191.2 Kb (ftp)(http) TXT
GSE127752_crisprTRIP_multiTRIP-GEMP-O_additionalHR3_3593_4_sgRNA2+ssODN_1uM_indels.txt.gz 208.4 Kb (ftp)(http) TXT
GSE127752_multiTRIP_GEMP-O_3109_mapping_normalization_expression_counts.txt.gz 156.2 Kb (ftp)(http) TXT
GSE127752_re-analysis_GEO_metadata_CRISPR-TRIP.xls.gz 34.7 Kb (ftp)(http) XLS
GSE127752_re-analysis_TRIP_cellline_EStTA-PB-B-18_mapping_normalization_expression_counts.txt.gz 1014 b (ftp)(http) TXT
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