Genome Editing
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Simple model for point mutation correction uses ssDNA repair oligo and CRISPR-Cas9 RNP

Rivera-Torres N, Banas K, et al. (2017) Insertional mutagenesis by CRISPR/Cas9 ribonucleoprotein gene editing in cells targeted for point mutation repair directed by short single-stranded DNA oligonucleotides. PLoS ONE, 12(1):e0169350. Doi:10.1371/journal.pone.0169350.

Background

CRISPR-Cas9 technology has been rapidly evolving as a method for gene editing and repair. However, protocols still require procedural improvements that increase editing precision and avoid “collateral mutagenesis” near the target site, and nonspecific editing at unintended genomic sites. In this research report, scientists in Dr Eric Kmiec‘s laboratory demonstrate the advantages of using a CRISPR-Cas9 ribonucleoprotein (RNP) format for DNA cleavage along with single-stranded DNA (ssDNA) oligonucleotides for direct nucleotide exchange at precise positions. The method results in repair of single-base mutations without accompanying insertions and/or deletions. To investigate the mechanism of repair by these gene editing tools, the group examined the genotype of individually sorted, corrected and uncorrected cells after clonal expansion.

Experiment

For this study, the Kmiec lab used HCT 116 cells containing a single-copy eGFP gene that has a stop codon (TAG) mutation in place of a tyrosine codon (TAC). Using the IDT Alt-R® CRISPR-Cas9 System in conjunction with a repair oligonucleotide, the researchers were able to convert the mutant stop codon to the normal tyrosine. Successful point mutation corrections resulted in functional, detectable eGFP. Following the repair process, the researchers clonally expanded targeted cells with and without a corrected eGFP gene. They then characterized DNA cleavage and repair events, and assessed the cell populations for collateral mutagenesis damage.

Results

The team used genotypic and phenotypic readout of a functional and detectable eGFP to assess point mutation correction. Employing the Alt-R CRISPR-Cas9 System and ssDNA repair oligo resulted in significant targeted gene correction of the mutant base (10–12%, reproducibly).

The authors state that use of an RNP format “delivers the active components of the CRISPR/Cas9 system to the nucleus at approximately the same time, facilitating a more constant initialization of the gene editing reaction.” They assert their studies show that point mutation is repair driven by the combination of the RNP and the ssDNA, as opposed to prior use of a plasmid expression system in which Cas9 is expressed from the same plasmid as the sgRNA.

The group also examined the targeted region of sorted, clonally expanded, cell populations containing the corrected and uncorrected gene (based on eGFP expression) to evaluate any collateral mutagenesis left by the action of the gene editing tools.

DNA sequence analysis of the eGFP expressing cells showed exact point mutation repair, with no adjacent sequence modification. However, similar analysis of cell populations containing an uncorrected gene revealed frequent collateral mutagenesis, with deletions and insertions surrounding the target site. In addition, 2 clonal populations that did not express eGFP did in fact show point mutation repair, but also included collateral mutagenesis near the target site. This latter result led the authors to emphasize the importance of analyzing mutagenicity in uncorrected cells.


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)
  • 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.


Alt-R CRISPR-Cpf1 System

The Alt-R CRISPR-Cpf1 System allows for new CRISPR target sites that are not available with the CRISPR-Cas9 System, and produces a staggered cut with a 5′ overhang. These reagents:

  • Enable genome editing in organisms with AT-rich genomes
  • Allow interrogation of additional genomic regions compared to Cas9
  • Require simply complexing the crRNA with the Cpf1 protein—no tracrRNA needed
  • Permit efficient delivery of the RNP into cells by electroporation

Learn more about the Alt-R CRISPR-Cpf1 System.


CRISPR support tools

Additional CRISPR reagents extend the ease-of-use and performance of the Alt-R system through options for fluorescent visualization, enhanced nuclease transfection, and genome editing detection.

Find out more about IDT’s entire line of CRISPR products.

Additional reading

User guide

Alt-R® CRISPR-Cas9 System User guide for cationic lipid delivery of CRISPR ribonucleoprotein into mammalian cells—Use the Alt-R S.p. Cas9 Nuclease 3NLS, in combination with the Alt-R CRISPR-Cas9 crRNA and tracrRNA, as a ribonucleoprotein (RNP) complex to achieve very high editing efficiency across most target sites. This guide discusses generation of the RNP using the Alt-R CRISPR-Cas9 System components and lipofection of the RNP into HEK-293 cells. Also, get general guidance for optimization of delivery in other cell types. Learn about analysis of editing results using T7 endonuclease I (T7EI) and how to use the Alt-R CRISPR-Cas9 System controls.

Additional protocols for the Alt-R CRISPR-Cas9 System are available here.


DECODED Online newsletter articles

Alt-R® CRISPR-Cas9 System ribonucleoprotein delivery optimization—Webinar: Genome editing using a Cas9:tracrRNA:crRNA ribonucleoprotein (RNP) provides excellent editing efficiency, while reducing off-target editing and cell death. Watch this recorded presentation to find out how to easily generate and deliver CRISPR RNAs and Cas9 nuclease in an RNP format, using the optimized Alt-R CRISPR-Cas9 System.

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.

Genome editing tip: A CRISPR-Cas9 RNA annealing step can increase editing efficiency—Looking for ways to increase the genome editing activity in your CRISPR experiments? This quick test suggests that spending a little extra time to anneal CRISPR RNAs will provide improvement.


Review other DECODED Online newsletter articles on CRISPR in genome editing applications.

You can also browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.


Author: Justin Barr, PhD, is the functional genomics product manager, and Ellen Prediger, PhD, is a senior scientific writer, both at IDT.

© 2017 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.


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Related Content

Product page:

Alt-R CRISPR-Cas9 System

Webinar:

New RNA tools for optimized CRISPR/Cas9 genome editing

Learn how research conducted at IDT led to the development of a potent new set of CRISPR-Cas9 genome editing tools.

Poster:

Quantitative Measurement of CRISPR/Cas9 Gene Editing at the Level of Genomic DNA for sgRNA Site Selection Algorithm Development

User guide:

Alt-R CRISPR-Cas9 System User Guide