citations

                    Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins
                

Authors:

Chen L, Park JE, Paa P, Rajakumar PD, Prekop HT, Chew YT, Manivannan SN, Chew WL.

In:

Source: Nat Commun.
Publication Date: (2021)
Issue: 12(1): 1384

Research Area:

Immunotherapy / Hematology

Cells used in publication:

H9 ( human ES cell line): Species: human; Tissue Origin: embryo

Platform:

4D-Nucleofector® X-Unit

Experiment

eHAP cells (Horizon Discovery, C669) were maintained in IMDM (Thermo Fisher, 31980-030) supplemented with 10% FBS at 37 °C and 5% CO2. 200,000 cells were nucleofected with 750 ng of base editor and 250 ng of gRNA expression plasmids using the SE Cell Line 4D-Nucleofector X Kit S (Lonza) and program DS-138 on the 4D X-Unit. Cells were harvested 72 h after nucleofection without any selection.

H9 stem cells (WiCell, WA09) were maintained in mTeSR1 (Stemcell technology, 85850). 200,000 cells were nucleofected with 1500 ng of base editor and 500 ng of gRNA expression plasmids using the P3 Primary Cell kit (Lonza, V4XP-3024) and program hES H9 program on the 4D X-Unit. Cells were harvested 72 h after nucleofection without any selection.

Abstract

Many genetic diseases are caused by single-nucleotide polymorphisms. Base editors can correct these mutations at single-nucleotide resolution, but until recently, only allowed for transition edits, addressing four out of twelve possible DNA base substitutions. Here, we develop a class of C:G to G:C Base Editors to create single-base genomic transversions in human cells. Our C:G to G:C Base Editors consist of a nickase-Cas9 fused to a cytidine deaminase and base excision repair proteins. Characterization of >30 base editor candidates reveal that they predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean 15.4% and up to 37% without selection). C:G to G:C Base Editors target cytidine in WCW, ACC or GCT sequence contexts and within a precise three-nucleotide window of the target protospacer. We further target genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of these base editors in interrogating and correcting human genetic diseases.

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