Project description:We confirmed Tyrosinase genome insertions or deletions of the three Tg mice by deep sequencing using MiSeq. These mice generated gene editing mice by using Tol2 transposon to introduce tyrosinase guide RNA (gRNA) into fertilized eggs obtained by crossing LSL (loxP-stop-loxP)-Cas9 mice with CAG-CreER mice.
Project description:RNA-Seq after Cas9-gRNA transfection with different length gRNAs we performed PolyA Selection and RNA-Seq on cells transfected with dCas9-VPR and a gRNA of each length (20nt, 16nt, or 14nt) targeting ACTC1, MIAT, or HBG1/2
Project description:The goal of these experiments were to test the on-target and target-adjacent editing efficiencies of different single-nucleobase editing systems. Previous studies have shown that tethering DNA mutating enzymes to Cas9-nickase-UGI complexes results in editing of chromosomal DNA. However, these editing events encompass undesirable target-adjacent nucleobase edits. Here, we characterize a novel approach that reduces the frequency of target-adjacent editing while maintaining a high level of on-target editing.
Project description:The CRISPR/Cas9 system shows diverse levels of genome editing activities on eukaryotic genomic DNA targets, and experiments desire high-efficiency targets. Here we show that chromatin open status is a pivotal determinant of the Cas9 editing activity in mammalian cells, and increasing chromatin accessibility can efficiently improve Cas9 genome editing activity. However, the strategy that fusing the VP64 transcriptional activation domain at the C-terminus of Cas9 can only promote genome editing activity slightly at most tested CRISPR/Cas9 targets in Lenti-X 293T cells. Because histone acetylation increases eukaryotic chromatin accessibility, we further improve genome editing by elevating histone acetylation. We demonstrate that promoting histone acetylation using histone acetyltransferase (HAT) activator YF-2 can improve genome editing from Cas9 and more robustly from the Cas9 transcriptional activator. This provides a strategy to improve CRISPR/Cas9 genome editing activity and enables broader gRNA target choices in eukaryotes.
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.
Project description:PolyA RNAseq of mouse ESC carrying a stable integration of an epigenetic editing machinery, consisting of: dCas9-GCN4, individual effectors fused to scFV and gRNA against the Hbby locus. The system is induced by doxycycline addition to the culture media. Total RNA was isolated from cell pellets using NEB Monarch® Total RNA Miniprep Kit and libraries prepared using NEBNext RNA Ultra II kit (NEB E7770S, input: 350 ng of total RNA). Libraries were multiplexed and sequenced on a NextSeq 500 (Paired-End; read length 40). Each sample is present in two biological replicates.
Project description:Structures of RNA editing substrate binding complexes reveal the mechanisms of guide RNA stabilization and messenger RNA recognition in the Trypanosoma brucei mitochondrion.
Project description:We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.