Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs

Okamoto, Sachiko; Amaishi, Yasunori; Maki, Izumi; Enoki, Tatsuji; Mineno, Junichi

doi:10.1038/s41598-019-41121-4

Cited by 113 publications

(127 citation statements)

References 37 publications

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“…The majority of KI animals created through electroporation of zygotes use single-stranded oligodeoxynucleotides (ssODN) donors, which have been shown to be stable and efficient in being incorporated into the genome through homology directed repair (HDR) 27 . Unfortunately, the size of the ssODN donor is a limitation as optimal ssODN donors are limited to a only about 100 base pairs 28 , with the largest successful insertion using this approach being 1kb in length 29,30 .…”

Section: Discussionmentioning

confidence: 99%

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

Owen

Hennig

McNabb

et al. 2020

Preprint

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11Introducing useful traits into livestock breeding programs through gene knock-ins has 12 proven challenging. Typically, targeted insertions have been performed in cell lines, followed by 13 somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce 14 genome editing reagents and a homologous recombination (HR) donor template into embryos to 15 trigger homology-directed repair (HDR). However, the HR pathway is primarily restricted to 16 actively dividing cells (S/G2-phase) and its efficiency is low in zygotes, especially for the 17 introduction of large DNA sequences. The homology-mediated end joining (HMEJ)-based 18 strategy harnesses HDR by direct injection of embryos, and has been shown to have an improved 19 knock-in efficiency in non-dividing cells. The knock-in efficiency for a 1.8kb gene was contrasted 20 when combining a gRNA/Cas9 ribonucleoprotein complex with either a traditional HR donor 21 template, or a HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly 22 higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). 23Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach 24 will facilitate the one-step introduction of gene constructs at a specific location of the bovine 25 genome and contribute to the next generation of elite cattle. 26 27Many attempts have been made to increase the rate of homologous recombination (HR) or 47 decrease the rate of non-homologous end joining (NHEJ) for gene insertion when using the 48 CRISPR/Cas9 system via CPI of zygotes 11 . However, these approaches have been unsuccessful in 49 bovine embryos as HR is primarily restricted to actively dividing cells 12 . Alternative homology 50 directed repair (HDR) approaches have been utilized for KI using a donor template via the 51 homology-mediated end joining (HMEJ) method 13 . This method has been shown to be active in 52 gametes and early stage 1-cell embryos, in which proteins necessary for pushing DNA repair 53 machinery towards the end-joining pathways are at their highest concentration 11 . While NHEJ 54 utilizes the Ku proteins and a ligase to mend the double-strand break (DSB) by blunt end ligation, 55 the HMEJ approach utilizes proteins for resection of the 5' ends and annealing of homologous 56 regions between the DSB and donor template similar to that in the microhomology-mediated end 57 joining (MMEJ) pathway 14 . 58In this study, we employed the HMEJ method for the precise insertion of the sex-59 determining region Y (SRY) gene into a region 10kb downstream of the zinc finger, X-linked (ZFX) 60 gene on the X chromosome of bovine embryos by injecting either in vitro matured oocytes prior 61 to in vitro fertilization, or presumptive zygotes 6hpi. We used two donor templates to compare KI 62 efficiency using the HMEJ and HR approaches, and show an increased rate of gene insertion at 63 the target location when using the HMEJ donor template. 64 RESULTS 65 Guide-RNA design and testing 66...

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Section: Discussionmentioning

confidence: 99%

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

Owen

Hennig

McNabb

et al. 2020

Preprint

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“…For the genome-editing, the DNA-directed RNA-guided endonuclease (RGEN) system (TakaraBio) was used [20]. Designed gRNA sequence was integrated into the expression vector under the U6 promotor (pRGEN_U6_SG).…”

Section: Genome-editing By Crispr/cas9mentioning

confidence: 99%

“…Regarding the method of introducing the Cas9, a transfection of the Cas9 proteins as a complex with gRNA (RNP) was used in the later experiment [20], instead of the standard expression vector method.…”

Section: Genome-editing By Crispr/cas9mentioning

confidence: 99%

Preparation of the standard cell lines for reference mutations in cancer gene-panels by genome editing in HEK 293 T/17 cells

et al. 2020

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Background: Next Generation Sequencer (NGS) is a powerful tool for a high-throughput sequencing of human genome. It is important to ensure reliability and sensitivity of the sequence data for a clinical use of the NGS. Various cancer-related gene panels such as Oncomine™ or NCC OncoPanel have been developed and used for clinical studies. Because these panels contain multiple genes, it is difficult to ensure the performance of mutation detection for every gene. In addition, various platforms of NGS are developed and their cross-platform validation has become necessity. In order to create mutant standards in a defined background, we have used CRISPR/Cas9 genome-editing system in HEK 293 T/17 cells. Results: Cancer-related genes that are frequently used in NGS-based cancer panels were selected as the target genes. Target mutations were selected based on their frequency reported in database, and clinical significance and on the applicability of CRISPR/Cas9 by considering distance from PAM site, and off-targets. We have successfully generated 88 hetero-and homozygous mutant cell lines at the targeted sites of 36 genes representing a total of 125 mutations. Conclusions: These knock-in HEK293T/17 cells can be used as the reference mutant standards with a steady and continuous supply for NGS-based cancer panel tests from the JCRB cell bank. In addition, these cell lines can provide a tool for the functional analysis of targeted mutations in cancer-related genes in the isogenic background.

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“…The majority of KI animals created through electroporation of zygotes use single-stranded oligodeoxynucleotides (ssODN) donors, which have been shown to be stable and efficient in being incorporated into the genome through homology directed repair (HDR) 31 . Unfortunately, the size of the ssODN donor is a limitation as optimal ssODN donors are limited to a only about 100 base pairs 32 . This limits donor repair templates to < 1 kb when electroporating ssODN 33 , although large insertions have been achieved in zygotes when using a ssODN-mediated KI approach in combination with microinjection of large donor repair templates 34 .…”

Section: Toi Donormentioning

confidence: 99%

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

Owen

Hennig

McNabb

et al. 2020

Sci Rep

View full text Add to dashboard Cite

Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Typically, targeted insertions have been performed in cell lines, followed by somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce genome editing reagents and a homologous recombination (HR) donor template into embryos to trigger homology directed repair (HDR). However, the HR pathway is primarily restricted to actively dividing cells (S/G2-phase) and its efficiency for the introduction of large DNA sequences in zygotes is low. The homology-mediated end joining (HMEJ) approach has been shown to improve knock-in efficiency in non-dividing cells and to harness HDR after direct injection of embryos. The knock-in efficiency for a 1.8 kb gene was contrasted when combining microinjection of a gRNA/Cas9 ribonucleoprotein complex with a traditional HR donor template or an HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach will facilitate the one-step introduction of gene constructs at a specific location of the bovine genome and contribute to the next generation of elite cattle.

show abstract

Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs

Cited by 113 publications

References 37 publications

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

Preparation of the standard cell lines for reference mutations in cancer gene-panels by genome editing in HEK 293 T/17 cells

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

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