Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing

doi:10.3390/ijms22168571

Review

. 2021 Aug 9;22(16):8571.

doi: 10.3390/ijms22168571.

Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing

Christopher E Denes¹, Alexander J Cole^{2

3}, Yagiz Alp Aksoy^{4

5}, Geng Li¹, Graham Gregory Neely^{1

2}, Daniel Hesselson^{2

3}

Affiliations

¹ The Dr. John and Anne Chong Lab for Functional Genomics, Charles Perkins Centre and School of Life & Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia.
² Centenary Institute, The University of Sydney, Sydney, NSW 2006, Australia.
³ Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia.
⁴ Sydney Medical School, The University of Sydney, Sydney, NSW 2006, Australia.
⁵ Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2113, Australia.

PMID: 34445274
PMCID: PMC8395304
DOI: 10.3390/ijms22168571

Review

Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing

Christopher E Denes et al. Int J Mol Sci. 2021.

. 2021 Aug 9;22(16):8571.

doi: 10.3390/ijms22168571.

Authors

Christopher E Denes¹, Alexander J Cole^{2

3}, Yagiz Alp Aksoy^{4

5}, Geng Li¹, Graham Gregory Neely^{1

2}, Daniel Hesselson^{2

3}

Affiliations

¹ The Dr. John and Anne Chong Lab for Functional Genomics, Charles Perkins Centre and School of Life & Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia.
² Centenary Institute, The University of Sydney, Sydney, NSW 2006, Australia.
³ Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia.
⁴ Sydney Medical School, The University of Sydney, Sydney, NSW 2006, Australia.
⁵ Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2113, Australia.

PMID: 34445274
PMCID: PMC8395304
DOI: 10.3390/ijms22168571

Abstract

Modification of the human genome has immense potential for preventing or treating disease. Modern genome editing techniques based on CRISPR/Cas9 show great promise for altering disease-relevant genes. The efficacy of precision editing at CRISPR/Cas9-induced double-strand breaks is dependent on the relative activities of nuclear DNA repair pathways, including the homology-directed repair and error-prone non-homologous end-joining pathways. The competition between multiple DNA repair pathways generates mosaic and/or therapeutically undesirable editing outcomes. Importantly, genetic models have validated key DNA repair pathways as druggable _targets for increasing editing efficacy. In this review, we highlight approaches that can be used to achieve the desired genome modification, including the latest progress using small molecule modulators and engineered CRISPR/Cas proteins to enhance precision editing.

Keywords: CRISPR/Cas9; engineered Cas9; genome editing; homology-directed repair; small molecules.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Figure 1**
Major DNA repair pathways. Double-stranded breaks are repaired by the error-prone non-homologous end joining (NHEJ) or the precise homology-directed repair (HDR) pathways.

**Figure 2**
Approaches for enhancing precision editing. Manipulations that favor HDR over NHEJ or which increase the accessibility of the genomic _target can increase editing efficacy.

**Figure 3**
Engineering Cas9 to expand the range of applications and precision of genome editing. *Streptococcus pyogenes* (Sp) Cas9 has been modified to reduce _target requirements and increase specificity. Partially inactivated (nCas9) and fully inactivated (dCas9) variants have been used to _target DNA-modifying activities to specific genomic loci.

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References

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[1] Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–821. doi: 10.1126/science.1225829. - DOI - PMC - PubMed

[2] Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–821. doi: 10.1126/science.1225829. - DOI - PMC - PubMed

[3] Jiang F., Zhou K., Ma L., Gressel S., Doudna J.A. STRUCTURAL BIOLOGY. A Cas9-guide RNA complex preorganized for _target DNA recognition. Science. 2015;348:1477–1481. doi: 10.1126/science.aab1452. - DOI - PubMed

[4] Jiang F., Zhou K., Ma L., Gressel S., Doudna J.A. STRUCTURAL BIOLOGY. A Cas9-guide RNA complex preorganized for _target DNA recognition. Science. 2015;348:1477–1481. doi: 10.1126/science.aab1452. - DOI - PubMed

[5] Hsu P.D., Lander E.S., Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157:1262–1278. doi: 10.1016/j.cell.2014.05.010. - DOI - PMC - PubMed

[6] Hsu P.D., Lander E.S., Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157:1262–1278. doi: 10.1016/j.cell.2014.05.010. - DOI - PMC - PubMed

[7] Sanjana N.E., Shalem O., Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods. 2014;11:783–784. doi: 10.1038/nmeth.3047. - DOI - PMC - PubMed

[8] Sanjana N.E., Shalem O., Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods. 2014;11:783–784. doi: 10.1038/nmeth.3047. - DOI - PMC - PubMed

[9] Gilbert L.A., Horlbeck M.A., Adamson B., Villalta J.E., Chen Y., Whitehead E.H., Guimaraes C., Panning B., Ploegh H.L., Bassik M.C., et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014;159:647–661. doi: 10.1016/j.cell.2014.09.029. - DOI - PMC - PubMed

[10] Gilbert L.A., Horlbeck M.A., Adamson B., Villalta J.E., Chen Y., Whitehead E.H., Guimaraes C., Panning B., Ploegh H.L., Bassik M.C., et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014;159:647–661. doi: 10.1016/j.cell.2014.09.029. - DOI - PMC - PubMed

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Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing

Affiliations

Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing

Authors

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Abstract

Conflict of interest statement

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