Project description:High-throughput sequencing of life stages and tissues of the flatworm Schistosoma mansoni to be used for further gene editing, gene finding and transcriptome analysis
Project description:High-fidelity Cas enzyme is pivotal to the safety of CRISPR technology in translational research and clinics. However, increasing editing fidelity often comes at the cost of a significant decrease in nuclease editing efficiency, e.g., the rationale designed SuperFi-Cas9 based on the structural basis for mismatch surveillance of wildtype Cas9. By conducting high-throughput profiling on the editing efficiency of 91,603 gRNAs, we found that SuperFi-Cas9/gRNA showed a strong nucleotide preference at the 21st position of the PAM-distal region of the target. A deep-learning model trained from the profiling data further provided a mutation map, which implied that a gRNA-target duplex with twenty-one matched nucleotides positively contributed to the editing efficiency of SuperFi-Cas9/gRNA. By further measuring the cleavage activities at twenty endogenous genomic locations, we demonstrated that a gRNA with extended 5' nucleotides significantly increased the editing activity of SuperFi-Cas9 while remaining its high fidelity, which makes SuperFi-Cas9 a valuable nuclease of the CRISPR toolbox. Together, by deep-learning modeling on high throughput profiling data, we reported that gRNAs with extended 5' nucleotides can rescue the impaired cleavage activities of SuperFi-Cas9.
Project description:A Scalable Epitope Tagging Approach for High Throughput ChIP-seq Analysis ChIP-seq comparison between CRISPR editing cells using epitope antibody and non-editing cells using endogeneous TF antibody
Project description:Because of their smallness, the recently developed CRISPR-Cas12f nucleases can be effectively packaged into adeno-associated viruses for gene therapy. However, a systematic evaluation of the editing outcomes of CRISPR-Cas12f is lacking. In this study, we apply a high-throughput sequencing method to comprehensively assess the editing efficiency, specificity, and safety of four Cas12f proteins in parallel with that of Cas9 and two Cas12a proteins at multiple genomic sites. Cas12f nucleases achieve robust cleavage at most of the tested sites and mainly produce deletional fragments. In contrast, Cas9 and Cas12a show relatively higher editing efficiency at the vast majority of the tested sites. However, the off-target hotspots identified in the Cas9- and Cas12a-edited cells are negligibly detected in the Cas12f-edited cells. Moreover, compared to Cas9 and Cas12a nucleases, Cas12f nucleases reduce the levels of chromosomal translocations, large deletions, and integrated vectors by 2- to 3-fold. Therefore, our findings confirm the editing capacity of Cas12f and reveal the ability of this nuclease family to preserve genome integrity during genome editing.
Project description:In E. coli, editing efficiency (EE) with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA DNA double-strand break (DSB)-induced cell death. We found that EE with the same gRNA and repair template can also change with target position, cas9 promoter strength, and growth conditions. Incomplete editing, off-target activity, non-targeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise EE. These effects on EE were gRNA-specific. We propose that differences in the efficiency of Cas9:gRNA-mediated DNA DSBs and differences in rates of dissociation of Cas9:gRNA complexes from target sites account for the observed variations in EE between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations.
2020-06-06 | GSE132139 | GEO
Project description:Systematic High Throughput Evaluation Reveals FrCas9 Superior Specificity and Efficiency for Therapeutic Genome Editing
Project description:High specificity of e\ngineered nucleases ensures precise genome editing. Couple methods were developed to identify off-target sites of CRISPR/Cas9, but hardly any high-throughput sequencing method can unequivocally determine their targeting efficiencies. Here we describe a comprehensive method, primer-extension-mediated sequencing (PEM-seq), which could sensitively detect CRISPR/Cas9 off-target sites as well as assess their editing efficiency by quantifying DNA interference events at on-target sites. Demonstrated by PEM-seq, we generated a high-fidelity Cas9 variant FeCas9 that possesses similar targeting ability as the wild-type while with extremely low off-target activities. Moreover, we provided further evidences for the broader range of xCas9 protospacer adjacent sequence. We also found the AcrIIA4 inhibitor could inhibit both on- and off-target activities of SpCas9, but it suppressed SpCas9 cleavage at the off-target loci not so efficiently as at the on-target sites. Finally, we believe PEM-seq is applicable to optimizing genome editing strategy for clinical purpose or creating animal model.