The use of base editing technology to characterize single nucleotide variants

Abstract

Single nucleotide variants (SNVs) represent the most common type of polymorphism in the human genome. However, in many cases the phenotypic impacts of such variants are not well understood. Intriguingly, while some SNVs cause debilitating diseases, other variants in the same gene may have no, or limited, impact. The mechanisms underlying these complex patterns are difficult to study at scale. Additionally, current data and research is mainly focused on European populations, and the mechanisms underlying genetic traits in other populations are poorly studied. Novel technologies may be able to mitigate this disparity and improve the applicability of personalized healthcare to underserved populations. In this review we discuss base editing technologies and their potential to accelerate progress in this field, particularly in combination with single-cell RNA sequencing. We further explore how base editing screens can help link SNVs to distinct disease phenotypes. We then highlight several studies that take advantage of single-cell RNA sequencing and CRISPR screens to emphasize the current limitations and future potential of this technique. Lastly, we consider the use of such approaches to potentially accelerate the study of genetic mechanisms in non-European populations.

Keywords: Base editing; CRISPR; Screens; Single nucleotide variants; Single-cell RNA sequencing.

Graphical abstract

Fig. 1

ClinVar distribution of SNV effects in humans and demonstration of the GWAS population biases. a. Graphs demonstrating the approximate percent of identified (by sequencing) human genetic variants that have been clinically classified (left), and the distribution of phenotypic effects, as listed in the ClinVar database (right). b. Estimated ratios of the GWAS in different populations during the last 15 years (the colors on the right represent the different populations). The plot was obtained from https://gwasdiversitymonitor.com/.

Fig. 2

Structure and mechanism of the CRISPR/Cas system. The CRISPR/Cas system (exemplified by Cas9 above) is composed of an sgRNA (shown in orange), which is complementary to the target DNA and binds to the target DNA, and a Cas protein (shown in grey), which helps bind and cleave the DNA through two nucleolytic domains. The HNH nuclease domain cleaves the complementary (i.e., target) strand while the RuvC nuclease cleaves the non-target strand. By definition, the non-target strand is called the protospacer, and has the same sequence as the sgRNA. The protospacer adjacent motif (PAM) is located directly downstream of the protospacer and is required for the Cas protein to initiate DNA binding . (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Schematic of the structural makeup and mechanism of base editors. Top: Cytosine base editors (CBEs) are composed of a catalytically impaired Cas9 protein (Cas9n) with a cytidine deaminase fused to the N-terminus and a uracil glycosylase inhibitor fused to the C-terminus. After the Cas9:sgRNA complex binds to the target DNA, the cytidine deaminase may convert any cytidine bases within the edit window to uridines. DNA repair pathways then preferentially replace the non-edited strand and incorporate an adenosine base across from the uridine. Overall, CBEs catalyze a C•G to T•A base pair conversion. Bottom: Adenine base editors (ABEs) are similar to CBEs but have an adenosine deaminase as the DNA base modifying enzyme and have no DNA repair inhibitor on the C-terminus. After the Cas9:sgRNA complex binds, adenosine residues in the edit window may be converted to inosines, which have the base-pairing properties of guanosine. Overall, ABEs catalyze an A•T to G•C base pair conversion.

Fig. 4

A general schematic of BE screens and single cell RNA sequencing (scRNAseq) workflows. Note, as there are multiple approaches that can be used for linking scRNAseq with BEs (or CRIPSRko/a/i) screens we provide here a general overview of the main steps. a. A library of sgRNA spacer sequences is designed and generated, then assembled into a viral vector. Lentiviruses are then produced, which are transduced into a population of target cells that express a BE. Expression of both the BE and a sgRNA will result in the introduction of an SNV of interest. The resulting cell population (which harbors a library of SNVs) is then subjected to a challenge to induce growth competition, followed by investigation of the effect of each SNV. b. After subjecting a pool of cells (that harbor a library of mutations) to a perturbation, the cells are passed through a microfluidics device to isolate individual cells into droplets containing a barcoded bead. The cells are lysed, and mRNA is captured by oligonucleotides on the beads. The oligonucleotides are reverse transcribed into a library, the droplets are recombined, then the library is sequenced and analyzed .