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Using CRISPR genome editing for gene knockout and homology-directed repair

Webinar review: Watch our webinar recording for expert guidance on a complete CRISPR genome editing workflow, including available tools and protocols. Also, see what we have learned about homology-directed repair and a new option for repair templates.

Apr 13, 2017

CRISPR has become an increasingly popular tool for targeted genome editing, because it is easier to implement and more robust than previously established methods, such as zinc-finger nucleases or TAL effector nucleases (TALENs). However, the wide range of products and methods available for CRISPR genome editing can also be daunting to sort through.

In the recorded presentation Getting started with CRISPR: a review of gene knockout and homology-directed repair, Justin Barr draws on our experimental data to guide you through the CRISPR workflow and available protocols. The presentation starts with a walkthrough of CRISPR RNA design using 2 popular, online tools: the CRISPR track that is built into the UCSC Genome Browser (https://genome.ucsc.edu) and the popular CRISPR design tool from the Broad Institute (http://crispr.mit.edu). Following the design discussion, Justin presents data that explains the advantages of a ribonucleoprotein (RNP) delivery format over other methods, and covers the components necessary to generate Cas9 RNPs.

A portion of the webinar also focuses on our investigation of the factors affecting homology-directed repair (HDR), including a brief introduction to very long, single-stranded DNA that can be used as an HDR template for inserting sequences into genomic sites. While either single- or double-stranded DNA can be used, single-stranded DNA has a higher HDR success rate. Single-stranded HDR templates up to 200 bases are readily available for small insertions or sequence replacement, but obtaining single-stranded DNA longer than 200 bases through chemical synthesis has been limiting factor for large insertions. To increase genome editing efficiency and reduce mutation screening, a proprietary manufacturing approach will soon allow IDT to make high quality, single-stranded DNA up to 2000 bases.

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