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 trypanosomatid flagellates possess in their single mitochondrion a highly complex kinetoplast (k) DNA, which is composed of interlocked circular molecules of two types. Dozens of maxicircles represent a classical mitochondrial genome, and thousands of minicircles encode guide (g)RNAs, which direct the processive and essential uridine insertion/deletion mRNA editing of maxicircle transcripts. While the details of kDNA structure and this type of RNA editing are well-established, our knowledge mostly relies on a narrow foray of intensely studied human parasites of the genera Leishmania and Trypanosoma. Here, we analyzed kDNA, its expression, and RNA editing of two members of the poorly characterized genus Vickermania with very different cultivation histories. In both Vickermania species, the gRNA-containing HL-circles are atypically large with multiple gRNAs each. Examination of V. spadyakhi HL-circle loci revealed a massive redundancy of gRNAs relative to the editing needs. In comparison, the HL-circle repertoire of extensively cultivated V. ingenoplastis is greatly reduced. It correlates with V. ingenoplastis-specific loss of productive editing of transcripts encoding subunits of respiratory chain complex I and corresponding lack of complex I activity. This loss in a parasite already lacking genes for subunits of complexes III and IV suggests an apparent requirement for its mitochondrial ATP synthase to work in reverse to maintain membrane potential. In contrast, V. spadyakhi retains a functional complex I that allows ATP synthase to work in its standard direction.
Project description:There is currently a lack of tools capable of perturbing genes in both a precise and spatiotemporal fashion. The flexibility of CRISPR, coupled with light's unparalleled spatiotemporal resolution deliverable from a controllable source, makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here we present a new optogenetic CRISPR tool (BLU-VIPR), that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of gRNA production. We engineered BLU-VIPR around a new potent blue-light activated transcription factor and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single mRNA transcript This simplified spatiotemporal gene perturbation and allowed for several types of optogenetic CRISPR, including indels, CRISPRa and base editing. BLU-VIPR also worked in vivo with cells previously intractable to optogenetic gene editing, achieving optogenetic gene editing in T lymphocytes in vivo.
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.