High-purity production and precise editing of DNA base editing ribonucleoproteins

Abstract

Ribonucleoprotein (RNP) complex–mediated base editing is expected to be greatly beneficial because of its reduced off-target effects compared to plasmid- or viral vector–mediated gene editing, especially in therapeutic applications. However, production of recombinant cytosine base editors (CBEs) or adenine base editors (ABEs) with ample yield and high purity in bacterial systems is challenging. Here, we obtained highly purified CBE/ABE proteins from a human cell expression system and showed that CBE/ABE RNPs exhibited different editing patterns (i.e., less conversion ratio of multiple bases to single base) compared to plasmid-encoded CBE/ABE, mainly because of the limited life span of RNPs in cells. Furthermore, we found that off-target effects in both DNA and RNA were greatly reduced for ABE RNPs compared to plasmid-encoded ABE. We ultimately applied NG PAM–targetable ABE RNPs to in vivo gene correction in retinal degeneration 12 (rd12) model mice.

Multiplex base editing to convert TAG into TAA codons in the human genome

“…However, a single guide RNA was designed for the highest homology among LINE-1 elements, which we predict generates a lower off-target mutation burden compared to multiple gRNAs. In the future, off-target mutations can be ameliorated with less off-target CBE 20,44 , RNP 45 and DddA-split base editors 46 .…”

Basic Principles and Clinical Applications of CRISPR-Based Genome Editing

“…The rd12 mouse model of LCA2 was subjected to subretinal injection of two AAVs encoding 1) SpCas9 and 2) sgRNA and donor DNA, resulting in the recovery of retinal function. 38 Recently, the same mouse model was successfully treated with subretinal injection of adenine base editors using RNPs 39 or intein-mediated split AAV vectors, 40 and prime editors using trans-splicing split AAV vectors, 41 showing promise in therapeutic genome editing with new genome editors.…”

Improvements of nuclease and nickase gene modification techniques for the treatment of genetic diseases

“…Furthermore, the product purity is also associated with the frequencies of Indels which are formed during the gene modifying process. Other studies have shown that over-expression of Uracil DNA glycosylase inhibitor (UGI) increased the purity of BE products in human cells ( Komor et al, 2017 ; Jiang et al, 2018 ; Jang et al, 2021 ) In addition to UGI, Gehrke et al (2018) used an engineered human enzyme known as APOBEC3A (eA3A) to develop the eA3A-BE3 base editor which improved target accuracy and reduced bystander mutations. To reduce the Indel formations, the BE4-Gam and CBEmax base editors have been developed ( Komor et al, 2017 ; Huang et al, 2019 ).…”