Title:
METHOD FOR REDUCING OFF-TARGET RATE OF CRISPR-CAS12A SPECIFICALLY CLEAVING TARGET NUCLEIC ACID BY CHANGING IONS
Document Type and Number:
WIPO Patent Application WO/2024/016730
Kind Code:
A1
Abstract:
A method for reducing the off-target rate of a Cas protein (such as CRISPR-Cas12a) specifically cleaving a target nucleic acid by changing the concentration of ions or ions. Specifically, provided is a reaction system comprising a solution of divalent metal ions, a Cas protein, and a guide RNA. The divalent metal ions comprise Ca2+ and non-calcium ions; the final concentration of Ca2+ in the solution is 0.1-200 mM, preferably 0.1-50 mM, and more preferably 0.1-10 mM; the final concentration of the non-calcium ions is lower than the final concentration of Ca2+; and the final concentration ratio of the non-calcium ions to Ca2+ is less than or equal to 99%, preferably less than or equal to 50%, more preferably less than or equal to 30%, more preferably less than or equal to 0.1%, and more preferably 0. The reaction system can remarkably reduce the trans-cleaving activity of the Cas protein but does not affect or enhance the cis-cleaving activity of the Cas protein, and can reduce the off-target rate of specifically cleaving a target nucleic acid.
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Inventors:
YIN LEI (CN)
WANG JIN (CN)
WANG JIN (CN)
Application Number:
PCT/CN2023/086002
Publication Date:
January 25, 2024
Filing Date:
April 03, 2023
Export Citation:
Assignee:
SHANGHAI TOLO BIOTECHNOLOGY COMPANY LTD (CN)
International Classes:
C12N9/22; C12N15/09; C12N15/113; C12Q1/6816
Foreign References:
| US20190048340A1 | 2019-02-14 | |||
| CN114507716A | 2022-05-17 | |||
| CN114088952A | 2022-02-25 | |||
| CN113373264A | 2021-09-10 |
Other References:
COFSKY JOSHUA C, KARANDUR DEEPTI, HUANG CAROLYN J, WITTE ISAAC P, KURIYAN JOHN, DOUDNA JENNIFER A: "CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks", ELIFE, vol. 9, 10 June 2020 (2020-06-10), XP093011978, DOI: 10.7554/eLife.55143
KIM HAN SEONG, KIM DONGWOOK, CHOE SUNGHWA: "Two novel mgCas12a proteins from the human gut metagenome exhibit reduced collateral DNase activity", RESEARCH SQUARE, 6 July 2022 (2022-07-06), XP093131677, [retrieved on 20240215], DOI: 10.21203/rs.3.rs-1720912/v1
KIM HAN SEONG, KIM DONGWOOK, CHOE SUNGHWA: "Two novel mgCas12a proteins from the human gut metagenome exhibit reduced collateral DNase activity", RESEARCH SQUARE, 6 July 2022 (2022-07-06), XP093131677, [retrieved on 20240215], DOI: 10.21203/rs.3.rs-1720912/v1
Attorney, Agent or Firm:
XU & PARTNERS, LLC. et al. (CN)
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