TY - JOUR

T1 - Natural Nucleoside Modifications in Guide RNAs Can Modulate the Activity of the CRISPR-Cas9 System In Vitro.

AU - Prokhorova, Daria V.

AU - Vokhtantsev, Ivan P.

AU - Tolstova, Polina O.

AU - Zhuravlev, Evgenii S.

AU - Kulishova, Lilia M

AU - Zharkov, Dmitry O

AU - Stepanov, Grigory A

PY - 2022/12

Y1 - 2022/12

N2 - At the present time, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has been widely adopted as an efficient genomic editing tool. However, there are some actual problems such as the off-target effects, cytotoxicity, and immunogenicity. The incorporation of modifications into guide RNAs permits enhancing both the efficiency and the specificity of the CRISPR-Cas9 system. In this study, we demonstrate that the inclusion of N 6-methyladenosine, 5-methylcytidine, and pseudouridine in trans-activating RNA (tracrRNA) or in single guide RNA (sgRNA) enables efficient gene editing in vitro. We found that the complexes of modified guide RNAs with Cas9 protein promoted cleavage of the target short/long duplexes and plasmid substrates. In addition, the modified monomers in guide RNAs allow increasing the specificity of CRISPR-Cas9 system in vitro and promote diminishing both the immunostimulating and the cytotoxic effects of sgRNAs.

AB - At the present time, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has been widely adopted as an efficient genomic editing tool. However, there are some actual problems such as the off-target effects, cytotoxicity, and immunogenicity. The incorporation of modifications into guide RNAs permits enhancing both the efficiency and the specificity of the CRISPR-Cas9 system. In this study, we demonstrate that the inclusion of N 6-methyladenosine, 5-methylcytidine, and pseudouridine in trans-activating RNA (tracrRNA) or in single guide RNA (sgRNA) enables efficient gene editing in vitro. We found that the complexes of modified guide RNAs with Cas9 protein promoted cleavage of the target short/long duplexes and plasmid substrates. In addition, the modified monomers in guide RNAs allow increasing the specificity of CRISPR-Cas9 system in vitro and promote diminishing both the immunostimulating and the cytotoxic effects of sgRNAs.

KW - CRISPR-Associated Protein 9/genetics

KW - CRISPR-Cas Systems/genetics

KW - Gene Editing

KW - Nucleosides

KW - RNA, Guide, Kinetoplastida/genetics

UR - https://www.mendeley.com/catalogue/0df0888b-7101-3864-8085-a61c5367efe6/

U2 - 10.1089/crispr.2022.0069

DO - 10.1089/crispr.2022.0069

M3 - Article

C2 - 36350691

VL - 5

SP - 799

EP - 812

JO - The CRISPR journal

JF - The CRISPR journal

SN - 2573-1599

IS - 6

ER -