Biology subjects

Moriarity, B.

Publications and source records attributed to Moriarity, B..

3 recordsLinked to original sources

bioRxiv2018

GeneWeld: a method for efficient targeted integration directed by short homology

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand break in vivo. Our results suggest that the length of homology is not important in the design of knock-in vectors but rather how the homology is presented to a double strand break in the genome. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR and TALEN compatible systems.

genetics↗

bioRxiv2018

Highly Multiplexed Genome Engineering Using CRISPR/Cas9 gRNA Arrays

The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limiting due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.

bioengineering↗

bioRxiv2017

EditR: A novel base editing quantification software using Sanger sequencing

CRISPR/Cas9-Cytidine deaminase fusion enzymes - termed Base Editors - allow targeted editing of genomic deoxcytidine to deoxthymidine (C[->]T) without the need for double stranded break induction. Base editors represent a paradigm-shift in gene editing technology, due to their unprecedented efficiency to mediate targeted, single-base conversion; however, current analysis of base editing outcomes rely on methods that are either imprecise or expensive and time consuming. To overcome these limitations, we developed a simple, cost effective, and accurate program to measure base editing efficiency from fluorescence-based Sanger sequencing, termed EditR. We provide EditR as a free online tool or downloadable desktop application requiring a single Sanger sequencing file and guide RNA sequence (baseeditr.com). EditR is more accurate than enzymatic assays, and provides added insight to the position, type and efficiency of base editing. Collectively, we demonstrate that EditR is a robust, inexpensive tool that will facilitate the broad application of base editing technology, thereby fostering further innovation in this burgeoning field.

genetics↗