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Precise and Predictable CRISPR Chromosomal Rearrangement Editing Reveals Principles of Cas9-mediated Nucleotide Insertion

ABSTRACT: Precise and Predictable CRISPR Chromosomal Rearrangement Editing Reveals Principles of Cas9-mediated Nucleotide Insertion

PROVIDER: PRJNA453692 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7067560.fastq.gz	Fastqsanger.gz
	SRR7067561.fastq.gz	Fastqsanger.gz
	SRR7067562.fastq.gz	Fastqsanger.gz
	SRR7067563.fastq.gz	Fastqsanger.gz
	SRR7067564.fastq.gz	Fastqsanger.gz

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Precise and Predictable CRISPR Chromosomal Rearrangement Editing Reveals Principles of Cas9-mediated Nucleotide Insertion

Project description:Chromosomal rearrangements including large DNA-fragment inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate structural genome variations and developmental gene regulation, but little is known about the underlying mechanism. Here we report that disrupting CtIP or FANCD2, which is thought to function in NHEJ, enhances precise DNA-fragment deletion. In addition, by analyzing the inserted nucleotides at the junctions of DNA-fragment deletions, inversions, duplications, and characterizing the cleaved products, we find that Cas9 endonucleolytically cleaves the noncomplementary strand with a flexible scissile profile upstream of -3 position of the PAM site in vivo and in vitro, generating overhanged DSB ends. Moreover, we find that engineered Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DNA-fragment editing could be achieved by perturbing DNA repair genes and using appropriate PAM configurations. These findings have important implications regarding 3D chromatin folding and enhancer insulation during gene regulation.

2018-07-26 | GSE113698 | GEO

Project description:Precise and Predictable CRISPR Chromosomal Rearrangement Editing Reveals Principles of Cas9-mediated Nucleotide Insertion

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Project description:Precise and Predictable CRISPR Chromosomal Rearrangement Editing Reveals Principles of Cas9-mediated Nucleotide Insertion

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Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy

Project description:Adoptive transfer of T cells expressing a transgenic T cell receptor (TCR) has the potential to revolutionize immunotherapy of infectious diseases and cancer. However, the generation of defined TCR-transgenic T cell medicinal products with predictable in vivo function still poses a major challenge and limits broader and more successful application of this ‘living drug’. Here, by studying 51 different TCRs, we show that conventional genetic engineering by viral transduction leads to variable TCR expression and functionality as a result of variable transgene copy numbers and untargeted transgene integration. In contrast, CRISPR/Cas9-mediated TCR replacement enables defined, targeted TCR transgene insertion into the TCR gene locus. Thereby, T cell products display more homogenous TCR expression similar to physiological T cells. Importantly, increased T cell product homogeneity after targeted TCR gene editing correlates with predictable in vivo T cell responses, which represents a crucial aspect for clinical application in adoptive T cell immunotherapy.

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Project description:A quantitative view of cellular functions requires precise measures of the rates of biomolecule production, especially proteins-the direct effectors of biological processes. Here we present a genome-wide approach, based on ribosome profiling, for measuring absolute protein synthesis rates. The resultant E. coli dataset transforms our understanding of the extent to which protein synthesis is precisely controlled to optimize function and efficiency. For example, members of multi-protein complexes are made in precise proportion to their stoichiometry, whereas components of functional modules are produced differentially according to their hierarchical role. Estimates of absolute protein abundance also reveal principles used to optimize design. These include how the level of different types of transcription factors is optimized for rapid response, and how a metabolic pathway (methionine biosynthesis) balances production cost with activity requirements. More broadly, our studies reveal how general principles, important both for understanding natural systems and for synthesizing new ones, emerge from global quantitative analyses of protein synthesis. 4 samples of E. coli ribosome profiling and mRNA-seq, including biological replicates

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