Development and Characterization of a Modular CRISPR and RNA Aptamer Mediated Base Editing System

Abstract

Conventional CRISPR approaches for precision genome editing rely on the introduction of DNA double-strand breaks (DSB) and activation of homology-directed repair (HDR), which is inherently genotoxic and inefficient in somatic cells. The development of base editing (BE) systems that edit a target base without requiring generation of DSB or HDR offers an alternative. Here, we describe a novel BE system called Pin-point^TM that recruits a DNA base-modifying enzyme through an RNA aptamer within the gRNA molecule. Pin-point is capable of efficiently modifying base pairs in the human genome with precision and low on-target indel formation. This system can potentially be applied for correcting pathogenic mutations, installing premature stop codons in pathological genes, and introducing other types of genetic changes for basic research and therapeutic development.

FIG. 1.

Description of Pin-point™ base editing (BE) platform and proof of principle in prokaryotic cells. (A) Components of the Pin-point platform, from left to right: 1 Sequence targeting component dCas9 or Cas9 nickases; 2 Chimeric RNA scaffold containing a guide RNA motif (for sequence targeting; 2.1), CRISPR motif (for Cas9 binding; 2.2), and recruiting RNA aptamer motif (for recruiting the effector RNA binding protein fusion; 2.3); and 3 Fusion protein consisting of the effector cytidine deaminase (3.1) fused to an RNA aptamer ligand (3.2). (B) Schematic of our modular complex at the target sequence: a dCas9 or a Cas9 nickase binds to CRISPR RNA, the recruiting RNA aptamer recruits the effector module, forming an active complex capable of editing target C residues (red) on the unpaired DNA within the CRISPR R-loop. The protospacer adjacent motif (PAM) sequence is shown in green. (C) Rifampicin resistance determining region (RRDR) Cluster I region of the Escherichia coli rpoB gene, with PAM sequences shown in green. Blue arrows represent gRNA targeting sites. The RRDR protein sequence is shaded in gray. (D) Surviving bacterial colonies after treatment with AID/dCas9 targeted with the indicated gRNAs carrying one copy of MS2 (1 × MS2). (E) Quantification of the survival fraction of cells from experiments shown in (D), showing the mean and standard deviation of three independent experiments. (F) Sequencing analysis of the rpoB gene from untreated cells (top) and AID/dCas9 treatment with rpoB_TS4_1 × MS2 gRNA (bottom). The target C is indicated with black asterisk. This C1592→T mutation results in a S531F change in protein sequence—a mutation known to induce rifampicin resistance.^,

FIG. 2.

Correction of a loss of function mutation in human cells. (A) Non-fluorescent enhanced green fluorescent protein (nfEGFP) target region. The chromophore sequence is underlined with the mutant amino acid C shown in red. One gRNA targeting the non-template strand (NT1) is shown as a blue arrow, the PAM sequence is shown in green, and the target cytosine is shown in red. The corresponding protein sequence is shaded in gray. (B) Extrachromosomal nfEGFP editing. Panels show representative sections of plates under a fluorescence microscope after the indicated treatments. The bar graph shows flow cytometry analysis of treated cells. (C) Flow cytometry analysis of nf2.16 cells stably expressing the non-fluorescent EGFP mutant gene after electroporation with nfEGFP_NT1 gRNA and either AIDv1, AID-BE4, or BE4max. (D) Electroporated cells were subjected to Sanger sequencing, and BE efficiency for each treatment was measured using the EditR analytical tool. All AID-BE4 and BE4max treatments were carried out with a gRNA lacking MS2 aptamers. Error bars represent standard deviation of the mean from three biologically independent experiments.

FIG. 3.

Pin-point-mediated BE at endogenous loci. HEK293T cells were electroporated with the indicated constructs and subjected to Sanger sequencing, and the BE outcomes were measured using the EditR analytical tool. First-generation AIDv1 and A1v1 constructs were targeted to Site 2 (A), Site 3 (B), and Site 4 (C). Second-generation AIDv2 and A1v2 analyzed at Site 2 (D) and Site 4 (E); AID-BE4 and BE4max are included as positive controls, targeted with gRNAs lacking MS2 aptamers. (F) Schematic representation of the dual-aptamer BE approach. By harnessing orthologous RNA aptamers and their binding proteins (e.g., MS2-MCP and PP7-PCP), heterologous Pin-point effectors can be programmed to perform dual-aptamer BE interventions, such as simultaneous delivery of AID and APOBEC1 to different loci. (G) Individual cytidine deamination by AIDv2^PCP on Site 2 and A1v2 on Site 4. (H) Dual-aptamer BE was achieved by combining AIDv2^PCP targeting Site2 and A1v2 targeting Site 4. Error bars represent standard deviation of the mean from three biologically independent experiments.

FIG. 4.

Introduction of a stop codon in a GFP reporter gene and an endogenous site in human cells. (A) Schematic representation of the targeted EGFP region. One gRNA (blue arrow) was designed to induce a stop codon at residue Q157 (EGFP_TS1; target C is shown in red). The corresponding protein sequence is shown in gray. (B) HEK293T cells expressing an EGFP transgene (293_GFP) were electroporated with or without AIDv2/EGFP_TS1 to induce knockout by nonsense mutation. Panels show sections of plates under a fluorescence microscope. (C) Quantification by flow cytometry of GFP expression in cells from experiments shown in (B). (D) 293_GFP cells electroporated with AIDv2/EGFP_TS1 were subjected to Sanger sequencing, and BE efficiencies were measured using the EditR analytical tool. (E) Schematic representation of the target region of the endogenous PDCD1 gene. One gRNA (blue arrow) was designed to induce a stop codon at residue Q133 (PDCD1_TS1; target C is shown in red). The corresponding protein sequence is shown in gray. (F) K562 cells were electroporated with AIDv2/PDCD1_TS1 were subjected to Sanger sequencing, and BE efficiencies were analyzed using the EditR analytical tool. Error bars represent standard deviation of the mean from three biologically independent experiments.