(614c) A Crispr-Cas9 Enabled Efficient and Scalable Method for High Throughput Chip-Seq Analysis
A CRISPR-Cas9 Enabled Efficient and Scalable Method for High Throughput Chip-Seq Analysis
Genome-wide profiling for protein-DNA interactions is crucial to understand the transcriptional regulation networks. DNA-binding proteins play essential roles in many cellular processes to provide growing insights about cell developmental stages and disease states. Eukaryotic nuclear DNA interacts with multiple proteins, forming the complexes chromatins. Chromatins exist as intermediates between completely compacted chromosomes and nucleosomes. Chromatin immunoprecipitation (ChIP) enriches DNA fragments to bind a specific protein or a class of nucleosomes. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is a high throughput sequencing technique to detect the protein-DNA binding events and chemical modifications of histone proteins. Followed by recognition and direct sequencing, more comprehensive sequence information can be obtained in a high throughput fashion.
All organisms enable specific transcription regulation on transcriptional factors (TFs) to interact with regulatory regions. Eukaryotic TFs typically recognize short genomic sequence and the clustering motifs can be found multiple times in large genomes. ChIP-seq study on TFs will open a new avenue to discover new motifs as well as unravel genomic targets for potential therapeutics. The quality of any ChIP data is highly dependent on antibody. Specific antibodies are demanded for enrichment and the increase of signal contrast over background. However, commercial antibodies for TFs in human genome are very limited. To overcome this major limitation, we developed a new method for ChIP-Seq analysis by fusing the TFs with commonly recognized polypeptide protein tag, 3×FLAG-tag, to allow the track of TFs with antibody against FLAG sequence. Specifically, we combine the clustered regularly interspaced short palindromic repeats (CRISPRs) technology with microhomology mediated end joining (MMEJ) to insert an 3×FLAG-tag with screening markers to the ends of TFs. Current strategies for chromosomal integration typically rely on CRISPR mediated integration vis homologous recombination (HR) or non-homologous end joining (NHEJ). Scaling HR mediated integration on human genome is less feasible; and the reported NHEJ integration efficiency is low. MMEJ provides novel cellular repair mechanism with more precise integration over NHEJ. Using the CRISPR-MMEJ mediated tagging approach, we envision its power to improve and broaden ChIP-seq technology on mapping the human genome. This new ChIP-seq strategy can be applied to any TF targets in all organisms.