Patent
Cas9-dna targeting unit chimeras
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TLDR
In this article, a Cas9-DTU fusion protein was proposed to facilitate single-site nuclease gene editing precision within a human genome. But, this method is not suitable for single-genomic-site accuracy.Abstract:
The present invention provides a Cas9 platform to facilitate single-site nuclease gene editing precision within a human genome. For example, a Cas9 nuclease/DN A- targeting unit (Cas9-DTU) fusion protein precisely delivers a Cas9/sgRNA complex to a specific target site within the genome for subsequent sgRNA-dependent cleavage of an adjacent target sequence. Alternatively, attenuating Cas9 binding using mutations to the a protospacer adjacent motif (PAM) recognition domain makes Cas9 target site recognition dependent on the associated DTU, all while retaining Cas9's sgRNA-mediated DNA cleavage fidelity. Cas9-DTU fusion proteins have improved target site binding precision, greater nuclease activity, and a broader sequence targeting range than standard Cas9 systems. Existing Cas9 or sgRNA variants (e.g., truncated sgRNAs (tru-gRNAs), nickases and Fokl fusions) are compatible with these improvements to further reduce off-target cleavage. A robust, broadly applicable strategy is disclosed to impart Cas9 genome-editing systems with the single-genomic-site accuracy needed for safe, effective clinical application.read more
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References
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Journal ArticleDOI
Delivering the goods: viral and non-viral gene therapy systems and the inherent limits on cargo DNA and internal sequences.
Helen Atkinson,Ronald Chalmers +1 more
TL;DR: The use of viruses and the limitations of current approaches to gene therapy are reviewed, followed by a more detailed analysis of transposon length and the physical properties of internal sequences, which both affect transposition efficiency.
Journal ArticleDOI
Global analysis of Drosophila Cys2-His2 zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants
Metewo Selase Enuameh,Yuna Asriyan,Adam Richards,Ryan G. Christensen,Victoria L. Hall,Majid Kazemian,Cong Zhu,Hannah Pham,Qiong Cheng,Charles Blatti,Jessie A. Brasefield,Matthew D. Basciotta,Jianhong Ou,Joseph C. McNulty,Lihua Julie Zhu,Susan E. Celniker,Saurabh Sinha,Gary D. Stormo,Michael H. Brodsky,Scot A. Wolfe +19 more
TL;DR: Subsets of fingers from Drosophila fingers were characterized to define their orientation and register on their recognition sequences, thereby allowing us to define the recognition diversity within this finger set, and it is found that the characterized fingers can specify 47 of the 64 possible DNA triplets.
Journal ArticleDOI
An improved predictive recognition model for Cys2-His2 zinc finger proteins
Ankit Gupta,Ryan G. Christensen,Heather A. Bell,Mathew J. Goodwin,Ronak Y. Patel,Manishi Pandey,Metewo Selase Enuameh,Amy L. Rayla,Cong Zhu,Stacey Thibodeau-Beganny,Michael H. Brodsky,J. Keith Joung,Scot A. Wolfe,Gary D. Stormo +13 more
TL;DR: The DNA-binding specificities of 678 two-finger modules from both natural and artificial sources are used to construct a random forest-based predictive model for ZFP recognition and it is found that this recognition model outperforms previously described determinant-based recognition models forZFPs, and can successfully estimate the specificity of naturally occurring ZFPs with previously defined specificities.
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Compositions and methods for promoting homology directed repair
TL;DR: In this paper, Cas9 molecules are used to create a break in a genomic region of interest, and then the cell can be contacted with molecules that bring a template nucleic acid in close proximity to the break, under conditions that allow the cell to repair the break using the template.
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