- •Recognize the newest techniques in biomedical research.
- •Describe how these techniques can be utilized and their limitations.
- •Describe the potential impact of these techniques.
- •CRISPR-Cas9 genome editing allows for precise gene repair or permanent gene knockout.
- •The CRISPR sgRNA honing mechanism makes genome-editing technology much easier to develop and produce compared with traditional designer nucleases.
- •CRISPR sgRNAs allow for multiple genes to be targeted simultaneously.
- •CRISPR-Cas9 genome editing can be used for genome-wide screens to identify genes and mutations responsible for complex biological processes.
- •Genome editing allows for the development of transgenic animal disease models.
- •Genome-editing technologies show promise as a therapeutic tool—for example, for treating genetic blistering diseases and for use as a selective antimicrobial agent.
- •Genome-editing modules may cut at nontargeted sites, resulting in off-target mutations with potentially adverse consequences to the host.
- •sgRNA design is restricted to gene targets containing a protospacer adjacent motif (PAM) sequence.
- •sgRNA efficiencies can be variable.
- •Homology-directed repair efficiency can be low.
- •Current genome-editing studies for treatment of recessive dystrophic epidermolysis bullosa (RDEB) rely on induced pluripotent stem cells (iPSCs), which can acquire mutations during reprogramming and form tumors if transplanted into patients.
Programmable Designer Nucleases for Genome Editing
Strategies of Genome Editing
Application of Genome Editing in Dermatological Research
Challenges and Future Directions
Multiple Choice Questions
- 1.A major advantage of CRISPR over traditional programmable designer nucleases is
- A.CRISPR’s reliance on sgRNA as the targeting mechanism.
- B.CRISPR’s higher gene-targeting efficiency.
- C.CRISPR’s lower off-targeting efficiency.
- 2.HDR is less efficient than NHEJ because
- A.HDR is inherently mutagenic and bad for the host.
- B.HDR relies on endogenous cellular proteins involved in homologous recombination.
- C.HDR relies on particular sequence-specific motifs that reduce its frequency, whereas NHEJ does not.
- 3.Off-target mutations are a result of
- A.mutant nuclease variants that randomly cut and mutate the genome.
- B.flawed sgRNA design, which hyperactivates Cas9 nuclease activity to randomly cut and mutate nontargeted sites.
- C.nontargeted sites sharing sequence homology with the targeted locus.
- 4.CRISPR-mediated genome-wide screens rely on ________ to target _______.
- A.sgRNA libraries; multiple genes simultaneously
- B.lentiviruses; gene mutations involved in the acquired resistance to melanoma chemotherapy agents
- C.Cas9; and kill melanoma cells
- 5.Cas9 is guided to the targeted locus by
- A.endogenous genomic palindromic adjacent motifs.
Conflict of Interest
- Quiz and brief explanation of correct answers
- Teaching Slides
Addgene. Overview of CRISPR/Cas9, https://www.addgene.org/crispr/guide/; 2016 (accessed 18 June 2016).
- Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials.Nat Biotechnol. 2014; 32: 1146-1150
- DNA nicks promote efficient and safe targeted gene correction.PLoS One. 2011; 6: e23981
- Use of induced pluripotent stem cells in dermatological research.J Invest Dermatol. 2014; 134: 1-5
- RNA-dependent DNA endonuclease Cas9 of the CRISPR system: holy grail of genome editing?.Trends Microbiol. 2013; 21: 562-567
- High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects.Nature. 2016; 529: 490-495
- Engineered CRISPR-Cas9 nucleases with altered PAM specificities.Nature. 2015; 523: 481-485
- Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.Nature. 2015; 517: 583-588
- Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining.Nat Biotechnol. 2015; 33: 538-542
- Repairing without cutting: a safer alternative to gene correction?.Mol Ther. 2014; 22: 690-691
- Somatic correction of junctional epidermolysis bullosa by a highly recombinogenic AAV variant.Mol Ther. 2014; 22: 725-733
- TALEN-based gene correction for epidermolysis bullosa.Mol Ther. 2013; 21: 1151-1159
- Potential pitfalls of CRISPR/Cas9-mediated genome editing.FEBS J. 2015; 283: 1218-1231
- Potential of systemic allogeneic mesenchymal stromal cell therapy for children with recessive dystrophic epidermolysis bullosa.J Invest Dermatol. 2015; 135: 2319-2321
- Advances in therapeutic CRISPR/Cas9 genome editing.Transl Res. 2016; 168: 15-21
- Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa.Sci Transl Med. 2014; 6: 264ra163
- Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells.Science. 2013; 343: 84-87
- Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.New Engl J Med. 2014; 370: 901-910
- Editing the epigenome: technologies for programmable transcription and epigenetic modulation.Nat Methods. 2016; 13: 127-137
- Making the cut.Science. 2015; 350: 1456-1457
User LicenseElsevier user license |
For non-commercial purposes:
- Read, print & download
- Text & data mine
- Translate the article
- Reuse portions or extracts from the article in other works
- Redistribute or republish the final article
- Sell or re-use for commercial purposes
Elsevier's open access license policy