Project description:Cmtm4 Knockout mouse was constructed by CRISPR-Cas9 technology, and the KO mice showed male infertility. A proteomic anlaysis of sperm proteins was performed by comparing KO and WT mice.
Project description:DNA was isolated from Apcmin/+;KrasLSL-G12D/+;VillinCre;Lgr5DTReGFP (AKVL), Apcmin/+;KrasLSL-G12D/+;VillinCre;Lgr5DTReGFP;p53KO (AKVPL) and Apcmin/+;KrasLSL-G12D/+;VillinCre;Lgr5DTReGFP;p53KO,Smad4KO (AKVPSL) organoids as well as the spleen of the AKVL donor animal The "SAMPLE_ID" sample characteristic is a sample identifier internal to Genentech. Overall design: Whole exome sequencing of murine derived intestinal organoids containing increasing number of CRC driver mutations was performed to identify potential mutations in coding regions that were introduced by off-target binding of the CRISPR-Cas9 complex.
Project description:By a robust unbiased ChIP-seq approach, we demonstrated that CRISPR/Cas9 had crRNA-specific off-target binding activities in human genome. However, most of those binding off-targets could not be efficiently cleaved both in vivo and in vitro which suggested the cleavage off-target activity of CRISPR/Cas9 in human genome is very limited. We provided a valuable tool to further investigate the molecular mechanism of CRISPR/Cas9 and to optimize its in vivo targeting sgRNA binding sites were identified with ChipSeq by using GFP antibody (there are 2 replicates for egfa-t1 sgRNA,emx1 sgRNA and control without sgRNA in Hek293T cells, one egfa-t1 sgRNA,emx1 sgRNA and control without sgRNA in HeLaS3 cells)
Project description:We report the use of short biotinylated oligonucleotides for the targeted pulldown of potential CRISPR/Cas9 off-target loci in condition and control to measure the frequency of off target editing Overall design: NG Capture-C was used to investigate the Potential off-target CRISPR/Cas9 loci. NG Capture-C combines 3C library preparation with oligonucleotide capture for the desired viewpoint restriction fragments. Potential off-target CRISPR/Cas9 loci were identified using the Sanger tool (http://www.sanger.ac.uk/htgt/wge/find_off_targets_by_seq) and 70bp oligonucleotides designed for target-pulldown. Whole genome amplified (WGA) DNA from edited clones from two editing strategies was pooled and indexed. Indexed WGA DNA was then pulled down in two rounds of hybridization with biotinylated oligonucleotides and streptavadin beads to enrich for putative off-target editing loci.
Project description:Purpose: DNA methyltransferase 3A (DNMT3A) mediates de novo DNA methylation. Mutations in DNMT3A are associated with hematological malignancies, most frequently acute myeloid leukemia. DNMT3A mutations are hypothesized to establish a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. However, the mechanisms by which DNMT3A mutations contribute to leukemogenesis are not well-defined. Methods: Whole-exome sequencing of extracted DNA was performed to confirm DNMT3A mutations. Library construction, exon capture, and sequencing was performed by Otogenetics (Atlanta, GA, USA). In brief, paired-end libraries were generated using the Illumina TruSeq DNA sample preparation kit. Exons were enriched using the Agilent Human All Exon V5 (51 Mb) capture system. Illumina HiSeq2500 was used for sequencing with a paired-end sequencing length of 100-125 bp and approximately 70 million reads per sample. Results: the DNMT3A mutations induced by K562 cells were transfected with a plasmid encoding Cas9, a CRISPR guide-RNA (gRNA) targeting the DNMT3A gene, and green fluorescent protein (GFP) followed by single-cell sorting. GFP-positive single cell clones were genotyped to confirm the presence of DNMT3A mutations. MT2-MT5 were verified by analysis of whole-exome sequencing data, and WTblk and WT1 were again confirmed to have no mutations in the DNMT3A gene. Conclusions: CRISPR/Cas9 gene editing allowed the generation of DNMT3A-mutated K562 cells that may be used to model effects of DNMT3A mutations in human cells. Our findings implicate aberrant splicing and induction of genomic instability as potential mechanisms by which DNMT3A mutations might predispose to malignancy. Overall design: whole-exome sequencing of wild type (WT) and DNMT3A mutated K562 cell lines were generated by deep sequencing using Illumina HiSeq2500
Project description:CRISPR-Cas9 delivery by AAV holds promise for gene therapy but faces critical barriers due to its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multi-functional platform customizable for genome-editing, transcriptional regulation, and other previously impracticable AAV-CRISPR-Cas9 applications. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV-CRISPR-Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce detectable cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics mRNA-Seq from muscles (9 samples; 3 mice x 3 conditions) and lymph nodes (9 samples; 3 mice x 3 conditions).
Project description:Identifying putative transcription factor target genes by combining CRISPR/Cas9-based transcriptional activation with RNAseq in Drosophila S2R+ cells. This study focuses on the transcription factors Twist and Snail, singly and together. RNA from Drosophila cells following CRISPR/Cas9-based activation of Twist, Snail, or Twist and Snail together, compared with non-targeting sgRNA. Two biological replicates for each experiment
Project description:CHD8 (chromodomain helicase DNA binding protein 8), which codes for a member of the CHD family of ATP-dependent chromatin-remodeling factors, is the most commonly mutated gene in autism spectrum disorders (ASD) identified in exome-sequencing studies. Loss of function mutations in the gene have also been found in schizophrenia (SZ) and intellectual disabilities, and affects cancer cell proliferation. To better understanding the molecular links between CHD8 functions and ASD, we have applied the CRISPR/Cas9 technology to knockout (KO) one copy of CHD8 in induced pluripotent stem cells (iPSCs) and build cerebral organoids, a model for the developing telencephalon. RNA-seq was carried out on KO organoids (CHD8+/-) and isogenic controls (CHD8+/+). Differentially expressed genes (DEGs) revealed an enrichment of genes involved in neurogenesis, forebrain development, Wnt/β-catenin signaling and axonal guidance. The SZ and bipolar disorder (BD) candidate gene TCF4 was significantly upregulated. Our CHD8 KO DEGs were significantly overlapped with those found in a transcriptome analysis using cerebral organoids derived from a family with idiopathic ASD and another transcriptome study using iPS cell-derived neurons from patients with BD, a condition characterized in a subgroup of patients by dysregulated WNT/β-catenin signaling. Overall, the findings show that distinct ASD, SZ and BD candidate genes converge on common molecular targets - an important consideration for developing novel therapeutics in genetically heterogeneous complex traits. Overall design: iPSCs derived from a healthy subject were transduced with CRISPR/Cas9 vectors with single guide RNA sequences to target the N-terminal of CHD8 protein to generate truncated mutations. The CHD8+/- iPSC lines were used to generate cerebral organoids for RNA-seq analysis, together with samples prepared from the parental clones, for a total of 6 samples (two biological replicates of wild-type (WT) and 4 biological replicates of CHD8+/-).