GSE55887

GSE GEO

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Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease

Organism: Homo sapiens

Platform: GPL15520

Samples: 12

Experiment Types:

Genome binding/occupancy profiling by high throughput sequencing

Submitted: Mar 13 2014

Last Updated: May 15 2019

Status: Public on May 18 2014

Contact: Mazhar,,Adli (Uni)

Relations

BioProject: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA241244 SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRP040086

Summary

RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from ~10 to >1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide

Overall Design

1.sgRNA1-6 binding sites were identified with ChipSeq by using HA antibody (there are 2 replicates for sgRNA1-3, one sample for sgRNA4-6,one control without sgRNA) 2.PCR products which amplifies " off-target genomic sites" were deep sequenced in the presence of WT Cas9+sgRNA or WT Cas9 alone( unique adaptor was used for each sgRNA and mixed for multiplex run)

Analysis (5 steps)

View Data Processing

Processing steps for GSE55887

Illlumina MiSeq Sequencer was used for base calling.
Sequence reads were aligned to hg19 reference genome using Bowtie for HA-Chip data and Bowtie2 for deep sequencing data.
Two re-sequenced files : sgRNA2_1_Replicate1 and sgRNA3_1_Replicate1 were merged with sgRNA2_Replicate1 and sgRNA3_Replicate1 respectively
Peaks were called by using MACS14 tool for HA-ChIP (-c=Control_dCas9only -p=1e-6)
Further data processing steps have been described in the method sections.

Supplementary Files (1)

GSE55887_RAW.tar Download

GEO Samples (12)

GSM1347963 GSM1347964 GSM1347965 GSM1347966 GSM1347967 GSM1347968 GSM1347969 GSM1347970 GSM1347971 GSM1347972 GSM1347973 GSM1347974

Dataset Citations (1)

PubMed citation (PMID 24837660)

SRA Experiments (12) and Runs (14)

Total: 7407 MB

SRX488710 SRP040086 ChIP-Seq SINGLE

GSM1347963: sgRNA1-replica1; Homo sapiens; ChIP-Seq

Sample: SRS572258

BioProject: PRJNA241244

BioSample: SAMN02688921

Platform: ILLUMINA

Instrument: Illumina MiSeq

Organism: Homo sapiens

Sample attributes

source_name: Human Embryonic Kidney

cell line: HEK 293T

passage: 17-21

chip antibody: HA(abcam #9110)

Original files (1)

Human Embryonic Kidney

Runs (1)

Run	Spots	Bases	Size (MB)	Files	Link
SRR1191627	8721934	1248554225	585.32	sgRNA1_Replicate1.fastq.gz, SRR1191627, SRR1191627.sralite	SRA

SRX488711 SRP040086 ChIP-Seq SINGLE

GSM1347964: sgRNA2-replica1; Homo sapiens; ChIP-Seq

Sample: SRS572259

BioProject: PRJNA241244

BioSample: SAMN02688922

Platform: ILLUMINA

Instrument: Illumina MiSeq

Organism: Homo sapiens

Sample attributes

source_name: Human Embryonic Kidney

cell line: HEK 293T

passage: 17-21

chip antibody: HA(abcam #9110)

Original files (1)

Human Embryonic Kidney

Runs (2)

Run	Spots	Bases	Size (MB)	Files	Link
SRR1191628	8159196	1163212904	501.37	sgRNA2_Replicate1.fastq.gz, SRR1191628, SRR1191628.sralite	SRA
SRR1191629	4257784	607687204	286.7	sgRNA2_1_Replicate1.fastq.gz, SRR1191629, SRR1191629.sralite	SRA

SRX488712 SRP040086 ChIP-Seq SINGLE

GSM1347965: sgRNA3-replica1; Homo sapiens; ChIP-Seq

Sample: SRS572260

BioProject: PRJNA241244

BioSample: SAMN02688919

Platform: ILLUMINA

Instrument: Illumina MiSeq

Organism: Homo sapiens

Sample attributes

source_name: Human Embryonic Kidney

cell line: HEK 293T

passage: 17-21

chip antibody: HA(abcam #9110)

Original files (1)

Human Embryonic Kidney

Runs (2)

Run	Spots	Bases	Size (MB)	Files	Link
SRR1191630	7089535	969461534	459.45	SRR1191630, SRR1191630.sralite, sgRNA3_Replicate1.fastq.gz	SRA
SRR1191631	5377060	733829690	317.76	SRR1191631, SRR1191631.sralite, sgRNA3_1_Replicate1.fastq.gz	SRA

SRX488713 SRP040086 ChIP-Seq SINGLE

GSM1347966: sgRNA1-replica2; Homo sapiens; ChIP-Seq

Sample: SRS572261

BioProject: PRJNA241244

BioSample: SAMN02688924

Platform: ILLUMINA

Instrument: Illumina MiSeq

Organism: Homo sapiens

Sample attributes

source_name: Human Embryonic Kidney

cell line: HEK 293T

passage: 17-21

chip antibody: HA(abcam #9110)

Original files (1)

Human Embryonic Kidney

Runs (1)

Run	Spots	Bases	Size (MB)	Files	Link
SRR1191632	10612847	1526529379	752.85	SRR1191632, SRR1191632.sralite, sgRNA1_Replicate2.fastq.gz	SRA

SRX488714 SRP040086 ChIP-Seq SINGLE

GSM1347967: sgRNA2-replica2; Homo sapiens; ChIP-Seq

Sample: SRS572262

BioProject: PRJNA241244

BioSample: SAMN02688923

Platform: ILLUMINA

Instrument: Illumina MiSeq

Organism: Homo sapiens

Sample attributes

source_name: Human Embryonic Kidney

cell line: HEK 293T

passage: 17-21

chip antibody: HA(abcam #9110)

Original files (1)

Human Embryonic Kidney

Runs (1)

Run	Spots	Bases	Size (MB)	Files	Link
SRR1191633	10904022	1583647050	778.97	sgRNA2_Replicate2.fastq.gz, SRR1191633, SRR1191633.sralite	SRA

SRX488715 SRP040086 ChIP-Seq SINGLE

GSM1347968: sgRNA3-replica2; Homo sapiens; ChIP-Seq

Sample: SRS572263

BioProject: PRJNA241244

BioSample: SAMN02688920

Platform: ILLUMINA

Instrument: Illumina MiSeq

Organism: Homo sapiens

Sample attributes

source_name: Human Embryonic Kidney

cell line: HEK 293T

passage: 17-21

chip antibody: HA(abcam #9110)

Original files (1)

Human Embryonic Kidney

Runs (1)

Run	Spots	Bases	Size (MB)	Files	Link
SRR1191634	10475165	1518187033	744.13	SRR1191634, SRR1191634.sralite, sgRNA3_Replicate2.fastq.gz	SRA

SRX488716 SRP040086 ChIP-Seq SINGLE

GSM1347969: sgRNA4; Homo sapiens; ChIP-Seq

Sample: SRS572264

BioProject: PRJNA241244

BioSample: SAMN02688929

Platform: ILLUMINA

Instrument: Illumina MiSeq

Organism: Homo sapiens

Sample attributes

source_name: Human Embryonic Kidney

cell line: HEK 293T

passage: 17-21

chip antibody: HA(abcam #9110)

Original files (1)

Human Embryonic Kidney

Runs (1)

Run	Spots	Bases	Size (MB)	Files	Link
SRR1191635	8855209	1274666515	597.87	sgRNA4.fastq.gz, SRR1191635, SRR1191635.sralite	SRA

SRX488717 SRP040086 ChIP-Seq SINGLE

GSM1347970: sgRNA5; Homo sapiens; ChIP-Seq

Sample: SRS572265

BioProject: PRJNA241244

BioSample: SAMN02688925

Platform: ILLUMINA

Instrument: Illumina MiSeq

Organism: Homo sapiens

Sample attributes

source_name: Human Embryonic Kidney

cell line: HEK 293T

passage: 17-21

chip antibody: HA(abcam #9110)

Original files (1)

Human Embryonic Kidney

Runs (1)

Run	Spots	Bases	Size (MB)	Files	Link
SRR1191636	5993795	879019630	406.82	sgRNA5.fastq.gz, SRR1191636, SRR1191636.sralite	SRA

SRX488718 SRP040086 ChIP-Seq SINGLE

GSM1347971: sgRNA6; Homo sapiens; ChIP-Seq

Sample: SRS572267

BioProject: PRJNA241244

BioSample: SAMN02688926

Platform: ILLUMINA

Instrument: Illumina MiSeq

Organism: Homo sapiens

Sample attributes

source_name: Human Embryonic Kidney

cell line: HEK 293T

passage: 17-21

chip antibody: HA(abcam #9110)

Original files (1)

Human Embryonic Kidney

Runs (1)

Run	Spots	Bases	Size (MB)	Files	Link
SRR1191637	7513613	1096361856	513.87	sgRNA6.fastq.gz, SRR1191637, SRR1191637.sralite	SRA

SRX488719 SRP040086 ChIP-Seq SINGLE

GSM1347972: dCas9 only; Homo sapiens; ChIP-Seq

Sample: SRS572266

BioProject: PRJNA241244

BioSample: SAMN02688927

Platform: ILLUMINA

Instrument: Illumina MiSeq

Organism: Homo sapiens

Sample attributes

source_name: Human Embryonic Kidney

cell line: HEK 293T

passage: 17-21

chip antibody: HA(abcam #9110)

Original files (1)

Human Embryonic Kidney

Runs (1)

Run	Spots	Bases	Size (MB)	Files	Link
SRR1191638	6944465	1014960814	462.65	Control_dCas9only.fastq.gz, SRR1191638, SRR1191638.sralite	SRA

SRX488720 SRP040086 ChIP-Seq SINGLE

GSM1347973: WT Cas9+sgRNA; Homo sapiens; ChIP-Seq

Sample: SRS572268

BioProject: PRJNA241244

BioSample: SAMN02688928

Platform: ILLUMINA

Instrument: Illumina MiSeq

Organism: Homo sapiens

Sample attributes

source_name: Human Embryonic Kidney

cell line: HEK 293T

passage: 19-21

chip antibody: none

Original files (1)

Human Embryonic Kidney

Runs (1)

Run	Spots	Bases	Size (MB)	Files	Link
SRR1191639	5013513	1149241941	500.71	SRR1191639, WTCas9+sgRNA.bam, SRR1191639.sralite	SRA

SRX488721 SRP040086 ChIP-Seq SINGLE

GSM1347974: WT Cas9 only; Homo sapiens; ChIP-Seq

Sample: SRS572269

BioProject: PRJNA241244

BioSample: SAMN02688930

Platform: ILLUMINA

Instrument: Illumina MiSeq

Organism: Homo sapiens

Sample attributes

source_name: Human Embryonic Kidney

cell line: HEK 293T

passage: 19-21

chip antibody: none

Original files (1)

Human Embryonic Kidney

Runs (1)

Run	Spots	Bases	Size (MB)	Files	Link
SRR1191640	5010114	1195917668	498.22	WTCas9only.bam, SRR1191640.sralite	SRA

Linked Publications (1)

Crosstalk between CRISPR-Cas9 and the human transcriptome.

Nature communications · 2022

Data Files (14)

Accession	File Name	Stored Type	Size	Download
—	Control_dCas9only.fastq.gz	ChIP-Seq	462.6 MB	link
—	sgRNA1_Replicate1.fastq.gz	ChIP-Seq	585.3 MB	link
—	sgRNA2_1_Replicate1.fastq.gz	ChIP-Seq	286.7 MB	link
—	sgRNA2_Replicate1.fastq.gz	ChIP-Seq	501.4 MB	link
—	sgRNA2_Replicate2.fastq.gz	ChIP-Seq	779.0 MB	link
—	sgRNA4.fastq.gz	ChIP-Seq	597.9 MB	link
—	sgRNA5.fastq.gz	ChIP-Seq	406.8 MB	link
—	sgRNA6.fastq.gz	ChIP-Seq	513.9 MB	link
—	SRR1191630	ChIP-Seq	459.4 MB	link
—	SRR1191631	ChIP-Seq	317.8 MB	link
—	SRR1191632	ChIP-Seq	752.8 MB	link
—	SRR1191634	ChIP-Seq	744.1 MB	link
—	SRR1191639	ChIP-Seq	500.7 MB	link
—	WTCas9only.bam	ChIP-Seq	498.2 MB	link