Engineered circular guide RNAs boost CRISPR/Cas12a- and CRISPR/Cas13d-based DNA and RNA editing

Abstract

Background: The CRISPR/Cas12a and CRISPR/Cas13d systems are widely used for fundamental research and hold great potential for future clinical applications. However, the short half-life of guide RNAs (gRNAs), particularly free gRNAs without Cas nuclease binding, limits their editing efficiency and durability.

Results: Here, we engineer circular free gRNAs (cgRNAs) to increase their stability, and thus availability for Cas12a and Cas13d processing and loading, to boost editing. cgRNAs increases the efficiency of Cas12a-based transcription activators and genomic DNA cleavage by approximately 2.1- to 40.2-fold for single gene editing and 1.7- to 2.1-fold for multiplexed gene editing than their linear counterparts, without compromising specificity, across multiple sites and cell lines. Similarly, the RNA interference efficiency of Cas13d is increased by around 1.8-fold. In in vivo mouse liver, cgRNAs are more potent in activating gene expression and cleaving genomic DNA.

Conclusions: CgRNAs enable more efficient programmable DNA and RNA editing for Cas12a and Cas13d with broad applicability for fundamental research and gene therapy.

Keywords: Cas12a; Cas13d; DNA editing; Engineered circular gRNA; Gene activation; RNA editing; cgRNA.

Fig. 1

Circularization increases the stability of gRNAs in human cells. a Schematic of circular guide RNAs (cgRNAs). b Broccoli fluorescence revealed the abundance of circular RNAs in cells. HEK293T cells were transfected with indicated plasmids encoding linear or circular RNAs and live stained with DFHBI-1T 48 hours (hrs) after transfection. c Reverse transcription PCR (RT-PCR) revealed circularization of RNAs in cells. HEK293T cells were transfected with indicated plasmids encoding linear or circular RNAs, and RNA was harvested 72 hrs after transfection, followed by RT-PCR with indicated outward-facing primers. d Stability of circular gRNA in cells. HEK293T cells were treated with actinomycin D for 1, 3, 6, 9, 18 hrs starting at 24 hrs post-transfection with plasmids encoding linear or circular RNAs, and RNA was harvested for quantitative RT-PCR analysis. n = 3 independent experiments

Fig. 2

Circular guide RNAs increase the transcription efficiency of Cas12a-based activators. a cgRNA-directed gene activation in a dLbCas12a-p300 knock-in (KI) HEK293T cell line. b Time-course analyses of cgRNA-directed gene activation in the KI cells. c Dose-dependent analyses of cgRNA-directed gene activation in the dLbCas12a-p300 knock-in HEK293T cells. d Gene activation guided by cgRNAs with different linkers in the KI cells. e–g cgRNA-directed gene activation with a variant of dLbCas12a-based gene activators in HEK293T cells transiently co-transfected with indicated activator-encoded and gRNA-encoded plasmids. h The specificity of cgRNA-directed gene activation. Gene expression plot generated from RNA-seq data from the KI HEK293T cells transfected with U6 + 27 linear gRNAs or C-L7 cgRNAs targeting mNeonGreen (control) or IL1RN. R indicates Pearson’s correlation coefficient. The average of three biological replicates was shown. For a–g, quantitative RT-PCR revealed relative mRNA expression of IL1RN and HBG. Mean values are presented with S.D., n = 3 independent experiments. For each experiment, fold changes of mRNA expression in tested samples versus that in the U6 + 27 linear mNeonGreen gRNA were shown. *p <0.05, **p <0.01, ***p <0.001, one-way ANOVA test

Fig. 3

Circular guide RNAs increase the DNA cleavage efficiency of Cas12a. a Fluorescence-activated cell sorting (FACS) analyses of the mNeonGreen reporter cells 4 days after co-transfection with LbCas12a-P2A-mCherry and mNeonGreen-targeting-gRNA plasmids. b The cleavage efficiency was quantified by the cell ratio of mNeonGreen^‒ mCherry⁺ / mCherry⁺ in the FACS assays. n = 3 independent experiments. NT gRNA, non-targeting gRNA, which recognized no site in the human genome and transcriptome. ***p <0.001, one-way ANOVA test. c–f The efficiency and specificity of different gRNAs-directed DNA cleavage at 14 sites in a LbCas12a knock-in HEK293T cell line revealed by Tag-seq. The gRNA reference as well as the on-target and off-target sites was shown on the left, and sequencing read counts were shown to the right of each site (c). Efficiency comparison between different gRNAs (d). The total number of off-target sites detected for the 14 sites (e). Specificity index (value was calculated by the ratio of total on-target reads to the on-target reads plus the off-target reads within the 14 sites) (f)

Fig. 4

Circular guide RNAs increase the RNA cleavage efficiency of CasRx. a, b The RNA cleavage efficiency of RfxCas13d (CasRx) on the mNeonGreen reporter gene. FACS analyses of the mNeonGreen knock-in HEK293T cell line 48 hrs after co-transfection with CasRx-P2A-mCherry and mNeonGreen-targeting-gRNA plasmids (a). The cleavage efficiency was quantified by the cell ratio of mNeonGreen^‒ mCherry⁺ / mCherry⁺ and mean fluorescence intensity (MFI) of mNeonGreen in transfected cells (mCherry positive) in the FACS assays (b). c Relative degradation of mNeonGreen transcripts induced by CasRx with circular or linear gRNAs. The mRNA expression levels were determined by RT-PCR. d The RNA cleavage efficiency of CasRx on endogenous genes. HEK293T cells were co-transfected with CasRx-P2A-mCherry and gRNA plasmids, and mCherry⁺ cells were sorted out by FACS for RNA extraction and quantitative RT-PCR analyses. n = 3 independent experiments. NT, Non-targeting gRNA, which recognized no site in the human genome and transcriptome. e The specificity of cgRNA-directed gene degradation. Gene expression plot generated from RNA-seq data from HEK293T cells transfected with non-targeting cgRNA or Pre linear gRNAs or C-L1 cgRNAs targeting NF2. R indicates Pearson’s correlation coefficient. The average of three biological replicates was shown. For b,c, **p <0.01, ***p <0.001, one-way ANOVA test. For d, **p <0.01, ***p <0.001, Student’s t test

Fig. 5

Circular gRNAs enhance the activation efficiency of dLbCas12a-VPR in vivo. a Experiment design for dLbCas12a-VPR to activate Luciferase expression in mouse liver. gCtrl, control gRNA, which targeted mNeonGreen. HTVI, hydrodynamic tail vein injection. b Representative bioluminescence imaging results at day 4 for all the 8 groups of mice. c Quantification of bioluminescence imaging detected for a consecutive 8 days. *p <0.05, **p <0.01, ***p <0.001, Student’s t test

Fig. 6

Circular gRNAs enhance the DNA cleavage efficiency of LbCas12a in vivo. a Experiment design for LbCas12a-N57 to induce liver tumor in adult mice. gCtrl, control gRNA, which targeted mNeonGreen. HTVI, hydrodynamic tail vein injection. b Image analysis of mouse liver harvested 7 weeks after injection. c Quantification of liver weight and surface liver tumor nodules per mouse. n = 7. d Representative images of H&E and IHC staining of Ck19 in mouse liver tumors. Scale bar, 100 μm. e Sanger sequencing results and tracking of indels by decomposition (TIDE) analyses of tumor DNA for Pten and Trp53 targeted sites. Blue shadow denoted the gRNA recognizing sites. f Genomic PCR of targeted integration of KRAS^G12D donor in tumors. Primers for the 5′-junction and 3′-junction were R26–5-F/R26–5-R and R26–3-F/R26–3-R, respectively. g Verification of the presence of the Kras^G12D mutation in the tumors by Sanger sequencing. *p <0.05, **p <0.01, Student’s t test