Engineering and optimising deaminase fusions for genome editing

Abstract

Precise editing is essential for biomedical research and gene therapy. Yet, homology-directed genome modification is limited by the requirements for genomic lesions, homology donors and the endogenous DNA repair machinery. Here we engineered programmable cytidine deaminases and test if we could introduce site-specific cytidine to thymidine transitions in the absence of targeted genomic lesions. Our programmable deaminases effectively convert specific cytidines to thymidines with 13% efficiency in Escherichia coli and 2.5% in human cells. However, off-target deaminations were detected more than 150 bp away from the target site. Moreover, whole genome sequencing revealed that edited bacterial cells did not harbour chromosomal abnormalities but demonstrated elevated global cytidine deamination at deaminase intrinsic binding sites. Therefore programmable deaminases represent a promising genome editing tool in prokaryotes and eukaryotes. Future engineering is required to overcome the processivity and the intrinsic DNA binding affinity of deaminases for safer therapeutic applications.

Rewritable multi-event analog recording in bacterial and mammalian cells

“…Base editors efficiently introduce single C•G to T•A mutations at guide RNA-programmed loci in a wide variety of eukaryotic cells and organisms (15, 16, 29–34). Predictable, durable point mutation of genomic or plasmid DNA by base editing has the potential to serve as an ideal information carrier in synthetic memory devices (Fig.…”

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

“…To address these limitations, the BE system has been fused to an array of Cas9 variants with varied PAM-sequence specificities derived from S. pyogenes (‘NGG’), S. aureus (‘NNGRRT’), or evolved artificially (‘NGA’, VQR-Cas9; ‘NGAG’, EQR-Cas9; ‘NGCG’, VRER-Cas9; ‘NNNRRT’, SaKKH-Cas9), thus allowing the BE system to target 2200 of these C:T or G:A point mutations (Kim et al, 2017c; Komor et al, 2017b). The space of targetable mutations has been further expanded by the fusion of cytidine deaminases to other programmable DNA binding proteins such as zinc-fingers and TALENs, which are not restricted by PAM sites (Yang et al, 2016a). …”

CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons

“…As an alternative to NHEJ- and HDR-dependent genome editing, CRISPR-dependent editing strategies that entail direct modification of DNA bases have recently been developed (Hess et al, 2016; Komor et al, 2016; Ma et al, 2016; Nishida et al, 2016; Plosky, 2016; Yang et al, 2016). Distinct from standard CRISPR-Cas9-dependent genome editing, CRISPR-mediated base editing avoids DSB formation and displays reduced genome-wide off-targeting (Kim et al, 2017a).…”