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

Abstract

The past several years have seen an explosion in development of applications for the CRISPR-Cas9 system, from efficient genome editing, to high-throughput screening, to recruitment of a range of DNA and chromatin-modifying enzymes. While homology-directed repair (HDR) coupled with Cas9 nuclease cleavage has been used with great success to repair and re-write genomes, recently developed base-editing systems present a useful orthogonal strategy to engineer nucleotide substitutions. Base editing relies on recruitment of cytidine deaminases to introduce changes (rather than double-stranded breaks and donor templates) and offers potential improvements in efficiency while limiting damage and simplifying the delivery of editing machinery. At the same time, these systems enable novel mutagenesis strategies to introduce sequence diversity for engineering and discovery. Here, we review the different base-editing platforms, including their deaminase recruitment strategies and editing outcomes, and compare them to other CRISPR genome-editing technologies. Additionally, we discuss how these systems have been applied in therapeutic, engineering, and research settings. Lastly, we explore future directions of this emerging technology.

Figure 1. Molecular components and mechanisms for base editing

A) The molecular components of the Base editor (BE), Target-AID, Targeted AID-mediated Mutagenesis (TAM), and CRISPR-X technologies are shown. The BE3, YEE-BE3, and Target-AID systems employ the Cas9 D10A (purple) nickase along with a cytidine deaminase (red) and a uracil DNA glycosylase inhibitor (yellow). The BE4 systems also uses the Gam protein (pink). The TAM and CRISPR-X systems used dCas9 (green) to recruit variants of the deaminase AID (AIDx or MS2-AID*Δ). Below each technology, the targeting window is indicated. The increased spectrum of base substitutions that can be achieved with TAM and CRISPR-X is represented by the multi-color editing windows. B) For base editors, the mechanisms of DNA repair are critical for the eventual editing outcome. After the initial deamination (C to U) conversion, uracil DNA glycosylase can remove the base and perform error-free (correctly replacing U with C) or error-prone repair generating different substitutions (left side). Alternatively a uracil DNA glycosylase inhibitor (UGI) can be used to block base excision by Uracil N-Glycosidase (UNG). Nicking via a Cas9 nickase of the non-deaminated strand promotes long patch base excision repair where the deaminated strand is preferred for templated repair generating a U:A base pair that is then resolved as a T:A base pair. Upon formation of the abasic site, an AP lyase can remove the site forming a ssDNA break. This excision in conjunction with the Cas9 D10A can create a DSB. Addition of the bacteriophage protein Gam reduces the frequency of indel formation by directly binding the double strand break, which may select against cells with DSBs. Endogenous repair machinery is labeled in black and represented with black triangles. Exogenous components of the base editing tools are illustrated and labeled in color.

Figure 2. Applications of base editing technologies

Base editing has been applied in two main areas: precision editing and screening applications. A) For precision editing, corrective or beneficial conversion of cytidines to thymidines has been demonstrated in many systems. Multiple delivery targets and methods that have been used are shown (see also Table 1). Precision editing has also been used for agricultural engineering using agrobacterium or biolistic DNA particle bombardment to deliver base editors to tomato, maize, wheat, and rice plants. B) Diversifying base editors have been used to evolve and study protein function and structure. In these experiments, sgRNAs tiling a region are used to generate targeted mutations and the subsequent population of variants is screened for specific phenotypes (i.e. drug resistance/fluorescence), followed by targeted sequencing of the locus. To validate identified mutations, enriched variants are individually installed and drug resistance or the activity of the individual clones are validated. C) The precise base editing system can also be used for screening applications. Targeting the BE3 system to Arg, Gln, and Trp codons can edit each into stop codons, generating truncated gene products (CRISPR-STOP, iSTOP). This platform provides a potential alternative to Cas9 gene screens without inducing double strand breaks.