Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency

Da Eun Jang, Jae Young Lee, Jae Hoon Lee, Ok Jae Koo, Hee Sook Bae, Min Hee Jung, Ji Hyun Bae, Woo Sung Hwang, Yoo Jin Chang, Yoon Hoo Lee, Han Woong Lee, Su Cheong Yeom

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

The CRISPR/Cas9 system is widely applied in genome engineering due to its simplicity and versatility. Although this has revolutionized genome-editing technology, knockin animal generation via homology directed repair (HDR) is not as efficient as nonhomologous end-joining DNA-repair-dependent knockout. Although its double-strand break activity may vary, Cas9 derived from Streptococcus pyogenens allows robust design of single-guide RNAs (sgRNAs) within the target sequence; However, prescreening for different sgRNA activities delays the process of transgenic animal generation. To overcome this limitation, multiple sets of different sgRNAs were examined for their knockin efficiency. We discovered profound advantages associated with single-stranded oligo-donor-mediated HDR processes using overlapping sgRNAs (sharing at least five base pairs of the target sites) as compared with using non-overlapping sgRNAs for knock-in mouse generation. Studies utilizing cell lines revealed shorter sequence deletions near target mutations using overlapping sgRNAs as compared with those observed using non-overlapping sgRNAs, which may favor the HDR process. Using this simple method, we successfully generated several transgenic mouse lines harboring loxP insertions or single-nucleotide substitutions with a highly efficiency of 18-38%. Our results demonstrate a simple and efficient method for generating transgenic animals harboring foreign-sequence knockins or short-nucleotide substitutions by the use of overlapping sgRNAs.

Original languageEnglish
Article number16
JournalExperimental and Molecular Medicine
Volume50
Issue number4
DOIs
Publication statusPublished - 2018 Apr 1

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Guide RNA
Repair
DNA End-Joining Repair
Genetically Modified Animals
Animals
Substitution reactions
Nucleotides
Genes
Sequence Deletion
Streptococcus
Base Pairing
Joining
Transgenic Mice
Cells
Genome
Technology
Cell Line
Mutation
DNA

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Clinical Biochemistry

Cite this

Jang, D. E., Lee, J. Y., Lee, J. H., Koo, O. J., Bae, H. S., Jung, M. H., ... Yeom, S. C. (2018). Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency. Experimental and Molecular Medicine, 50(4), [16]. https://doi.org/10.1038/s12276-018-0037-x
Jang, Da Eun ; Lee, Jae Young ; Lee, Jae Hoon ; Koo, Ok Jae ; Bae, Hee Sook ; Jung, Min Hee ; Bae, Ji Hyun ; Hwang, Woo Sung ; Chang, Yoo Jin ; Lee, Yoon Hoo ; Lee, Han Woong ; Yeom, Su Cheong. / Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency. In: Experimental and Molecular Medicine. 2018 ; Vol. 50, No. 4.
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abstract = "The CRISPR/Cas9 system is widely applied in genome engineering due to its simplicity and versatility. Although this has revolutionized genome-editing technology, knockin animal generation via homology directed repair (HDR) is not as efficient as nonhomologous end-joining DNA-repair-dependent knockout. Although its double-strand break activity may vary, Cas9 derived from Streptococcus pyogenens allows robust design of single-guide RNAs (sgRNAs) within the target sequence; However, prescreening for different sgRNA activities delays the process of transgenic animal generation. To overcome this limitation, multiple sets of different sgRNAs were examined for their knockin efficiency. We discovered profound advantages associated with single-stranded oligo-donor-mediated HDR processes using overlapping sgRNAs (sharing at least five base pairs of the target sites) as compared with using non-overlapping sgRNAs for knock-in mouse generation. Studies utilizing cell lines revealed shorter sequence deletions near target mutations using overlapping sgRNAs as compared with those observed using non-overlapping sgRNAs, which may favor the HDR process. Using this simple method, we successfully generated several transgenic mouse lines harboring loxP insertions or single-nucleotide substitutions with a highly efficiency of 18-38{\%}. Our results demonstrate a simple and efficient method for generating transgenic animals harboring foreign-sequence knockins or short-nucleotide substitutions by the use of overlapping sgRNAs.",
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Jang, DE, Lee, JY, Lee, JH, Koo, OJ, Bae, HS, Jung, MH, Bae, JH, Hwang, WS, Chang, YJ, Lee, YH, Lee, HW & Yeom, SC 2018, 'Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency', Experimental and Molecular Medicine, vol. 50, no. 4, 16. https://doi.org/10.1038/s12276-018-0037-x

Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency. / Jang, Da Eun; Lee, Jae Young; Lee, Jae Hoon; Koo, Ok Jae; Bae, Hee Sook; Jung, Min Hee; Bae, Ji Hyun; Hwang, Woo Sung; Chang, Yoo Jin; Lee, Yoon Hoo; Lee, Han Woong; Yeom, Su Cheong.

In: Experimental and Molecular Medicine, Vol. 50, No. 4, 16, 01.04.2018.

Research output: Contribution to journalArticle

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T1 - Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency

AU - Jang, Da Eun

AU - Lee, Jae Young

AU - Lee, Jae Hoon

AU - Koo, Ok Jae

AU - Bae, Hee Sook

AU - Jung, Min Hee

AU - Bae, Ji Hyun

AU - Hwang, Woo Sung

AU - Chang, Yoo Jin

AU - Lee, Yoon Hoo

AU - Lee, Han Woong

AU - Yeom, Su Cheong

PY - 2018/4/1

Y1 - 2018/4/1

N2 - The CRISPR/Cas9 system is widely applied in genome engineering due to its simplicity and versatility. Although this has revolutionized genome-editing technology, knockin animal generation via homology directed repair (HDR) is not as efficient as nonhomologous end-joining DNA-repair-dependent knockout. Although its double-strand break activity may vary, Cas9 derived from Streptococcus pyogenens allows robust design of single-guide RNAs (sgRNAs) within the target sequence; However, prescreening for different sgRNA activities delays the process of transgenic animal generation. To overcome this limitation, multiple sets of different sgRNAs were examined for their knockin efficiency. We discovered profound advantages associated with single-stranded oligo-donor-mediated HDR processes using overlapping sgRNAs (sharing at least five base pairs of the target sites) as compared with using non-overlapping sgRNAs for knock-in mouse generation. Studies utilizing cell lines revealed shorter sequence deletions near target mutations using overlapping sgRNAs as compared with those observed using non-overlapping sgRNAs, which may favor the HDR process. Using this simple method, we successfully generated several transgenic mouse lines harboring loxP insertions or single-nucleotide substitutions with a highly efficiency of 18-38%. Our results demonstrate a simple and efficient method for generating transgenic animals harboring foreign-sequence knockins or short-nucleotide substitutions by the use of overlapping sgRNAs.

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