Author response: Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery
Open accessAbstract
Building on previous work (Jinek et al., 2013), we report a simple and robust system to achieve high fidelity and high efficiency (30% of homologous recombination) genome engineering by homology-directed repair pathway in human cells using cell cycle synchronization and timed delivery of Cas9 ribonucleoprotein complexes.
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Longer homologous arms and single-stranded oligonucleotide DNA templates, compared with double-strand DNA templates, can increase HDR rate.
“The authors also demonstrated that longer homologous arm and single-stranded oligonucleotide DNA template (compared with double-strand DNA templates) can increase HDR rate.”
From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [63]
Timed delivery of Cas9 protein and sgRNA complexes into human cells via cell cycle synchronisation can achieve an HDR rate up to 38%.
“Via cell cycle synchronisation techniques, Lin and colleagues 63 conducted timed delivery of Cas9 protein and sgRNA complexes into human cells and gained HDR rate up to 38%.”
From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [63]
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“DSBs in the genome provoke natural DNA repair systems such as non-homologous end joining (NHEJ), or homology-directed repair (HRD) in the cell, consequently introducing indels or precise insertions, respectively, at the target site in the host genome (Lin et al., 2014).”
BackgroundNon-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes
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“Donor DNA complementary to the non-target strand stimulated HDR frequencies and is more effective than donor complementary to the target strand (Lin et al. 2014; Richardson et al. 2016; Yang et al. 2013).”
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“Therefore, delivery of pre-transcribed gRNA and pre-translated Cas9 as Cas9/ Ribonucleoprotein (Cas9/RNP) has been favorable in immune cells (20, 21).”
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“In this setting, the challenges associated with transducing such a large protein can be circumvented through electroporation of Cas9 RNPs (58, 59).”
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“HDR, on the other hand, is preferentially used to repair DSBs that occur in the S–G2 phase of the cell cycle, when homologous DNA sequences from the sister chromatid are available as a template to repair the lesion.”
InfluentialDefining and targeting patterns of T cell dysfunction in inborn errors of immunity
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Enhancing CRISPR deletion via pharmacological delay of DNA-PKPreprint
Bosch, N., Medová, M., Esposito, R., Pulido-Quetglas, C., Zimmer, Y., & Johnson, R. (2020) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2020.02.12.945907
“…to boost efficiency are: (1) direct stimulation of homology directed repair (HDR) (Riesenberg and Maricic 2018; Yeh et al. 2019; Song et al. 2016; Lin et al. 2014); (2) suppression of the competing NHEJ pathway at early stages through inhibition of Ku70/80 complex (Fattah et al. 2008; Riesenberg…”
Background
- Authors
- Lin, S., Staahl, B. T., Alla, R. K., & Doudna, J. A.
- Affiliations
- University of California, Berkeley (US)
- QB3 (US)
- Howard Hughes Medical Institute (US)
- Venue
- eLife
- Publisher
- eLife Sciences Publications, Ltd
- Published
- 2014-12-04
- ISSN
- 2050-084X (ISSN-L)
- OpenAlex
- W2987080681
- Cited by
- 15
- References
- 0
- Citations by year
- 2025: 1 · 2023: 1 · 2022: 4 · 2021: 1 · 2020: 4 · 2019: 1 · 2017: 1 · 2016: 2
- Type
- peer review
- Language
- en
- Open access
- gold
- doi.org
- Locations
- eLife (publishedVersion, cc-by)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- homology-directed repair, CRISPR/Cas9, genome editing, cell cycle synchronization, Cas9 ribonucleoprotein, homologous recombination, human cells, genome editing efficiency
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