Bio-Orthogonal Chemistry Conjugation Strategy Facilitates Investigation of N-methyladenosine and Thiouridine Guide RNA Modifications on CRISPR Activity.
CRISPR J
; 5(6): 787-798, 2022 12.
Article
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| MEDLINE
| ID: mdl-36378256
ABSTRACT
The CRISPR-Cas9 system is an important genome editing tool that holds enormous potential toward the treatment of human genetic diseases. Clinical success of CRISPR technology is dependent on the incorporation of modifications into the single-guide RNA (sgRNA). However, chemical synthesis of modified sgRNAs, which are over 100 nucleotides in length, is difficult and low-yielding. We developed a conjugation strategy that utilized bio-orthogonal chemistry to efficiently assemble functional sgRNAs containing nucleobase modifications. The described approach entails the chemical synthesis of two shorter RNA oligonucleotides a 31-mer containing tetrazine (Tz) group and a 70-mer modified with a trans-cyclooctene (TCO) moiety. The two oligonucleotides were conjugated to form functional sgRNAs. The two-component conjugation methodology was utilized to synthesize a library of sgRNAs containing nucleobase modifications such as N1-methyladenosine (m1A), N6-methyladenosine (m6A), 2-thiouridine (s2U), and 4-thiouridine (s4U). The impact of these RNA modifications on overall CRISPR activity were investigated in vitro and in Cas9-expressing HEK293T cells.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Tiouridina
/
Edición Génica
Límite:
Humans
Idioma:
En
Año:
2022
Tipo del documento:
Article