Identification of functional sgRNA mutants lacking canonical secondary structure using high-throughput FACS screening.
Cell Rep
; 43(6): 114290, 2024 Jun 25.
Article
in En
| MEDLINE
| ID: mdl-38823012
ABSTRACT
Coexpressing multiple identical single guide RNAs (sgRNAs) in CRISPR-dependent engineering triggers genetic instability and phenotype loss. To provide sgRNA derivatives for efficient DNA digestion, we design a high-throughput digestion-activity-dependent positive screening strategy and astonishingly obtain functional nonrepetitive sgRNA mutants with up to 48 out of the 61 nucleotides mutated, and these nonrepetitive mutants completely lose canonical secondary sgRNA structure in simulation. Cas9-sgRNA complexes containing these noncanonical sgRNAs maintain wild-type level of digestion activities in vivo, indicating that the Cas9 protein is compatible with or is able to adjust the secondary structure of sgRNAs. Using these noncanonical sgRNAs, we achieve multiplex genetic engineering for gene knockout and base editing in microbial cell factories. Libraries of strains with rewired metabolism are constructed, and overproducers of isobutanol or 1,3-propanediol are identified by biosensor-based fluorescence-activated cell sorting (FACS). This work sheds light on the remarkable flexibility of the secondary structure of functional sgRNA.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Flow Cytometry
/
RNA, Guide, CRISPR-Cas Systems
Language:
En
Journal:
Cell Rep
Year:
2024
Document type:
Article
Affiliation country:
China
Country of publication:
Estados Unidos