Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells

Abstract

CRISPR-Cas9 is quickly revolutionizing the way we approach gene therapy. CRISPR-Cas9 is a complexed, two-component system using a short guide RNA (gRNA) sequence to direct the Cas9 endonuclease to the target site. Modifying the gRNA independent of the Cas9 protein confers ease and flexibility to improve the CRISPR-Cas9 system as a genome-editing tool. gRNAs have been engineered to improve the CRISPR system's overall stability, specificity, safety, and versatility. gRNAs have been modified to increase their stability to guard against nuclease degradation, thereby enhancing their efficiency. Additionally, guide specificity has been improved by limiting off-target editing. Synthetic gRNA has been shown to ameliorate inflammatory signaling caused by the CRISPR system, thereby limiting immunogenicity and toxicity in edited mammalian cells. Furthermore, through conjugation with exogenous donor DNA, engineered gRNAs have been shown to improve homology-directed repair (HDR) efficiency by ensuring donor proximity to the edited site. Lastly, synthetic gRNAs attached to fluorescent labels have been developed to enable highly specific nuclear staining and imaging, enabling mechanistic studies of chromosomal dynamics and genomic mapping. Continued work on chemical modification and optimization of synthetic gRNAs will undoubtedly lead to clinical and therapeutic benefits and, ultimately, routinely performed CRISPR-based therapies.

Keywords: CRISPR therapeutics; CRISPR-Cas9; chemical modifications; engineered nuclease; gRNA; gene therapy; genome editing.

Figure 1

Type II CRISPR formulations. gRNAs contain 4 loop structures: tetraloop (green), Stem-loop 1 (yellow), Stem-loop 2 (orange), and Stem-loop 3 (magenta). Stem-loop 2 and tetraloop do not interact with Cas9 as they protrude from the nuclease (Konermann et al., 2015). The spacer region of the guide undergoes Watson and Crick base pairing with the complimentary stand to the DNA protospacer. The spacer region (also known as guide region) is typically 20 nucleotides long but it has been shown that it can be shortened or lengthened (to include hairpin structures) at the 5′ end. The spacer region can be divided into two regions: the PAM-proximal (seed) region and the PAM-distal region. (A) Naturally occurring crRNA [~42 nt (striped nucleotides)] containing the DNA-binding spacer sequence and the trans-activating tracrRNA [80 nt (Rahdar et al., 2015) (checkered nucleotides)] annealed together through Watson and Crick base-pairing by the repeat (brown) and anti-repeat (gray) regions. (B) Synthetic sgRNA formulation where the crRNA and tracrRNA are covalently fused by a tetraloop. R-loop formation is depicted with Watson and Crick base pairing of the RNA:DNA heteroduplex.

Figure 2

Chemical modifications on the ribose rings and phosphate backbone of gRNAs. Ribose modifications are typically placed at the 2′OH as it is readily available for manipulation. Simple modifications at the 2′OH include 2′-O-Me, 2′-F, and 2′F-ANA. More extensive ribose modifications such as 2′F-4′-Cα-OMe and 2′,4′-di-Cα-OMe combine modification at both the 2′ and 4′ carbons. Phosphodiester modifications include sulfide-based Phosphorothioate (PS) or acetate-based phosphonoacetate alterations. Combinations of the ribose and phosphodiester modifications have given way to formulations such as 2′-O-methyl 3′phosphorothioate (MS), or 2′-O-methyl-3′-thioPACE (MSP), and 2′-O-methyl-3′-phosphonoacetate (MP) RNAs. Locked and unlocked nucleotides such as locked nucleic acid (LNA), bridged nucleic acids (BNA), S-constrained ethyl (cEt), and unlocked nucleic acid (UNA) are examples of sterically hindered nucleotide modifications. Modifications to make a phosphodiester bond between the 2′ and 5′ carbons (2′,5′-RNA) of adjacent RNAs as well as a butane 4-carbon chain link between adjacent RNAs have been described. ‘A’ symbolizes the nitrogen base of the RNA.

Figure 3

Various applications of engineered gRNAs. (A,B)–gRNA modifications to improve HDR: (A) crRNA-donor DNA conjugate. The donor DNA is fused to the 5′ end of the guide region. (B) sgRNA molecule with streptavidin-binding aptamers that attach to either the tetraloop or stem-loop 2 (the two loops protruding from the Cas9 molecule). The formulation has the donor ssODN bound to a biotin molecule that binds the streptavidin tightly to ensure the proximity of the donor DNA to the break site. (C,D)–gRNA modifications that utilize CRISPR-dCas9 specificity for high-resolution cellular imaging: (C) sgRNA molecule with fluorophore-bound aptamers binding to either the tetraloop or stem-loop 2 (for the same reason as mentioned above). GFP and BFP were shown solely as examples since CRISPRainbow covers the full spectrum of combinations. (D) CRISPR LiveFISH method utilizes crRNAs fused to a fluorophore at the 5′ end to actuate live intracellular staining without the need for cellular fixation. (E) Light-activated CRISPR to allow for control over synchronous editing across a cell population. Photocaging with light-sensitive 6′-nitropiperonyloxymethyl (NPOM) thymidine modifications on the distal portion of the guide region prevents the gRNA from binding completely to its DNA target. Following exposure to light, the NPOM modifications are released and complete binding and subsequent editing commence.