Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells
Open accessAbstract
CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34(+) hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.
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CRISPR-Cas9 enables programmable targeting of indels at a chosen genomic site.
“The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nuclease system, a bacterial immune system that can cleave bacteriophage or plasmid DNA, enables programmable targeting of insertions or deletions (indels) at a specific genomic DNA site.28,29”
From CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia (2021) · cites this paper as [29]
Experimental — accuracy not yet measuredThe modified sgRNAs carry 2'-O-methyl-3'-phosphorothioate changes at the three terminal nucleotides on both ends, as previously described.
“The modified sgRNAs contain 2’-O-methyl-3’-phosphorothioate modifications at the three terminal nucleotides of the 5’ and 3’ ends as described previously12.”
From A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells (2018) · cites this paper as [12]
Chemical modification of sgRNA improved gene editing in vitro when co-delivered with Cas9 mRNA.
“A recent study demonstrated that the chemical modification of sgRNA improved gene editing in vitro when co-delivered with Cas9 mRNA32.”
From Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo (2016) · cites this paper as [32]
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G-quadruplex-based CRISPR photoswitch for spatiotemporal control of genomic modulation
Deng, H., Xu, H., Wang, Y., Jia, R., Ma, X., Feng, Y., & Chen, H. (2023) · Nucleic Acids Research · doi:10.1093/nar/gkad178 · Cited by 45 · see citing works
Massively targeted evaluation of therapeutic CRISPR off-targets in cells
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Self-citationTechnical considerations for the use of CRISPR/Cas9 in hematology research
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Nanoblades allow high-level genome editing in murine and human organoids
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A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes
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“53 Some cell types, such as primary human natural killer (NK) cells and monocytes, may benefit from addition of RNase inhibitor (Millipore Sigma, Cat #3335399001) to reduce degradation of the mRNA and gRNA by endogenous RNases.”
- Authors
- Hendel, A., Bak, R. O., Clark, J. T., Kennedy, A. B., Ryan, D. E., Roy, S., Steinfeld, I., Lunstad, B. D., Kaiser, R. J., Wilkens, A. B., Bacchetta, R., Tsalenko, A., Dellinger, D., Bruhn, L., & Porteus, M. H.
- Affiliations
- Stanford Medicine (US)
- Agilent Technologies (United States) (US)
- Corresponding author
- Bruhn, Laurakay
- Porteus, Matthew H
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2015-09
- Volume
- 33
- Issue
- 9
- Pages
- 985-989
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC4729442
- OpenAlex
- W2209402024
- Cited by
- 1,090
- References
- 25
- Citations by year
- 2026: 62 · 2025: 80 · 2024: 100 · 2023: 106 · 2022: 127 · 2021: 113 · 2020: 141 · 2019: 93 · 2018: 101 · 2017: 74 · 2016: 73 · 2015: 18 · 2014: 1
- FWCI
- 38.48
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health (5r01ai120766-02)
- National Eye Institute (PN2EY018244)
- National Institute of Allergy and Infectious Diseases (R01-AI120766, R01AI097320)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- CRISPR/Cas genome editing, single guide RNA, RNA delivery, CD34+ hematopoietic stem and progenitor cells, genome editing efficiency
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Genetic Engineering, Humans, Genome, Human, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
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