Research Of CRISPR-mediated HDR Method And ChIP-seq Method

Clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated(Cas)proteins are derived from the adaptive immune system of bacteria against phages and other foreign genetic elements such as plasmid.Currently,the type II CRISPR/Cas9 system is widely used.Cas9 recognizes and cleaves genome-specific DNA sequences through base-pair pairing under sgRNA guide,resulting in genomic double-strand breaks,followed by introduction of specific site mutations by non-homologous end joining(NHEJ),or inserting or substituting a specific fragment into a specific site by either homologous directed repair(HDR)or microhomology-mediated end joining(MMEJ).The use of traditional CRISPR/Cas9 systems for HDR-mediated specific site repairing are inefficient and an efficient HDR-mediated precision repair method is highly desired.This study intended to increase the efficiency of Cas9-mediated HDR by providing a repair template near the Cas9 cleavage site.Avidin was covalent linked to Cas9 via the SpyTag-SpyCatcher system to form a Cas9-avidin protein complex.A DNA HDR template with biotin modification was pre-incubated with the Cas9-avidin protein complex before delivered into the cells.We designed an additional 15-nt short sgRNAs targeting the upstream and downstream of the Cas9 cleavage site respectively,which guides the Cas9-avidin protein complex with a repair template to the vicinity of the cleavage site.The results showed that the produced HDR efficiency was slightly improved in HEK293 cells with this system.At the same time,Gu et al.reported a similar method that increased the HDR efficiency to 95% in the mouse embryo at 2-cell stage and reduced the off-target rate effectively,proving this strategy is feasible.DNA-binding proteins are mainly involved in the maintenance of genome integrity,chromatin modification,gene expression regulation and other biological processes.ChIP-seq(Chromatin immunoprecipitation experiments followed by sequencing)is a powerful method for genome-wide studying of DNA/protein interactions.However,non-specific bindings of non-target proteins and DNA to antibodies and beads during immunoprecipitation result in higher false positive ratio.Herein,we have improved the ChIP-seq using the SpyTag-SpyCatcher covalent labeling system.By introducing a SpyTag tag to the protein of interest,the target DNA/protein complex can be pulled down through the spontaneous covalent cross-linking reaction between the SpyTag-fused protein and the SpyCatcher coupling with the magnetic beads.This covalent cross-linking is resistant to harsh washing conditions such as high concentration of SDS or boiling,which efficiently removed the non-specifically bound protein and DNA without the cost of losing the target DNA/protein complex.We named this technique as SpyChIP-seq.Our results showed that SpyChIP-seq can effectively enrich the target DNA/protein complex in HEK293 T cells stably expressing SpyTag-tagged transcription factors ELK4 and BACH1 with significantly improved signal-to-noise ratio.