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Series GSE113698 Query DataSets for GSE113698
Status Public on Jul 26, 2018
Title Precise and Predictable CRISPR Chromosomal Rearrangement Editing Reveals Principles of Cas9-mediated Nucleotide Insertion
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Summary Chromosomal rearrangements including large DNA-fragment inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate structural genome variations and developmental gene regulation, but little is known about the underlying mechanism. Here we report that disrupting CtIP or FANCD2, which is thought to function in NHEJ, enhances precise DNA-fragment deletion. In addition, by analyzing the inserted nucleotides at the junctions of DNA-fragment deletions, inversions, duplications, and characterizing the cleaved products, we find that Cas9 endonucleolytically cleaves the noncomplementary strand with a flexible scissile profile upstream of -3 position of the PAM site in vivo and in vitro, generating overhanged DSB ends. Moreover, we find that engineered Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DNA-fragment editing could be achieved by perturbing DNA repair genes and using appropriate PAM configurations. These findings have important implications regarding 3D chromatin folding and enhancer insulation during gene regulation.
 
Overall design HEK293T expression profiles of CtIP knockout
 
Contributor(s) Shou J, Li J, Liu Y, Wu Q
Citation(s) 30033371
Submission date Apr 26, 2018
Last update date Mar 20, 2019
Contact name Jia Shou
Organization name Shanghai Jiao Tong University
Department Institute of Systems Biomedicine
Lab Qiang Wu
Street address 800 Dongchuan Rd
City Shanghai
State/province Shanghai
ZIP/Postal code 200240
Country China
 
Platforms (1)
GPL20795 HiSeq X Ten (Homo sapiens)
Samples (6)
GSM3112281 WT rep1
GSM3112282 WT rep2
GSM3112283 CtIP-KO C14 rep1
Relations
BioProject PRJNA453692
SRA SRP142730

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Supplementary file Size Download File type/resource
GSE113698_RAW.tar 242.9 Mb (http)(custom) TAR (of BW)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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