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

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.

Product focus—genome editing with Alt-R® CRISPR Reagents

Alt-R CRISPR-Cas9 System

The Alt-R CRISPR-Cas9 System includes all the reagents needed for successful genome editing. Based on the natural S. pyogenes CRISPR-Cas9 system, the Alt-R CRISPR-Cas9 System offers numerous advantages over alternative methods:

  • Higher on-target potency than other CRISPR systems
  • Precision control with delivery of Cas9 ribonucleoprotein (RNP) complexes
  • Efficient delivery of the RNP with lipofection or electroporation
  • No toxicity or innate immune response activation, in contrast to in vitro transcribed Cas9 mRNA and sgRNAs

Learn more about the Alt-R CRISPR-Cas9 System.

Templates for homology-directed repair (HDR)—Ultramer® Oligonucleotides

Ultramer® Oligonucleotides are high-fidelity, single-stranded DNA sequences up to 200 nt. Have them custom synthesized to contain your insertion sequence and homology arms for use in CRISPR HDR experiments. 

Find out more at www.idtdna.com/pages/products/dna-rna/ultramer-oligos

Additional reading

CRISPR genome editing: 5 considerations for target site selection—Read how your genome editing experiments can be improved with 5 quick tips for target selection and with reagents from the Alt-R® CRISPR-Cas9 System.

Simple model for point mutation correction uses ssDNA repair oligo and CRISPR-Cas9 RNP—Citation summary: This publication demonstrates how CRISPR-Cas9 ribonucleoprotein (RNP) complexes (used for DNA cleavage), and ssDNA oligonucleotides (used for repair), will correct single-base mutations without collateral mutagenesis in the surrounding sequence. Read the authors' explanation for why this CRISPR reagent delivery format is so successful.

A simple method to detect on-target editing or measure genome editing efficiency in CRISPR experiments—Product spotlight: Learn why the Alt-R® Genome Editing Detection Kit, a PCR-based, T7 endonuclease I (T7EI) assay, is the recommended method for CRISPR mutation detection.

Review other DECODED Online newsletter articles on genome editing applications.

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Author: Hans Packer, PhD, is a scientific writer at IDT.

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