Rationally Designed Anti-CRISPR Nucleic Acid Inhibitors of CRISPR-Cas9.
Nucleic Acid Ther
; 29(3): 136-147, 2019 06.
Article
in En
| MEDLINE
| ID: mdl-30990769
ABSTRACT
Clustered regularly interspaced short palindromic repeat (CRISPR) RNAs and their associated effector (Cas) enzymes are being developed into promising therapeutics to treat disease. However, CRISPR-Cas enzymes might produce unwanted gene editing or dangerous side effects. Drug-like molecules that can inactivate CRISPR-Cas enzymes could help facilitate safer therapeutic development. Based on the requirement of guide RNA and target DNA interaction by Cas enzymes, we rationally designed small nucleic acid-based inhibitors (SNuBs) of Streptococcus pyogenes (Sp) Cas9. Inhibitors were initially designed as 2'-O-methyl-modified oligonucleotides that bound the CRISPR RNA guide sequence (anti-guide) or repeat sequence (anti-tracr), or DNA oligonucleotides that bound the protospacer adjacent motif (PAM)-interaction domain (anti-PAM) of SpCas9. Coupling anti-PAM and anti-tracr modules together was synergistic and resulted in high binding affinity and efficient inhibition of Cas9 DNA cleavage activity. Incorporating 2'F-RNA and locked nucleic acid nucleotides into the anti-tracr module resulted in greater inhibition as well as dose-dependent suppression of gene editing in human cells. CRISPR SNuBs provide a platform for rational design of CRISPR-Cas enzyme inhibitors that should translate to other CRISPR effector enzymes and enable better control over CRISPR-based applications.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
DNA-Binding Proteins
/
CRISPR-Cas Systems
/
Gene Editing
/
CRISPR-Associated Protein 9
Limits:
Humans
Language:
En
Journal:
Nucleic Acid Ther
Year:
2019
Document type:
Article