Evaluation of Engineered CRISPR-Cas-Mediated Systems for Site-Specific RNA Editing.
Ryan J Marina, Kristopher W Brannan, Kevin D Dong, Brian A Yee, Gene W Yeo
Cell reports · 2020 · Vol. 33 (5) · pp. 108350
Abstract
Site-directed RNA editing approaches offer great potential to correct genetic mutations in somatic cells while avoiding permanent off-target genomic edits. Nuclease-dead RNA-targeting CRISPR-Cas systems recruit functional effectors to RNA molecules in a programmable fashion. Here, we demonstrate a Streptococcus pyogenes Cas9-ADAR2 fusion system that uses a 3' modified guide RNA (gRNA) to enable adenosine-to-inosine (A-to-I) editing of specific bases on reporter and endogenously expressed mRNAs. Due to the sufficient nature of the 3' gRNA extension sequence, we observe that Cas9 gRNA spacer sequences are dispensable for directed RNA editing, revealing that Cas9 can act as an RNA-aptamer-binding protein. We demonstrate that Cas9-based A-to-I editing is comparable in on-target efficiency and off-target specificity with Cas13 RNA editing versions. This study provides a systematic benchmarking of RNA-targeting CRISPR-Cas designs for reversible nucleotide-level conversion at the transcriptome level.
Linked datasets 1
Discovery and evaluation of CRISPR/Cas-mediated systems for site-specific RNA editing
Homo sapiens
30 data files
| File | Type | Size |
|---|---|---|
| Cas13b_CYFIP2_gRNA_1_R1_001.fastq.gz | RNA-Seq | 4.2 GB |
| Cas13b_CYFIP2_gRNA_2_R1_001.fastq.gz | RNA-Seq | 2.7 GB |
| Cas13b_NT_gRNA_1_R1_001.fastq.gz | RNA-Seq | 4.3 GB |
| Cas13b_NT_gRNA_2_R1_001.fastq.gz | RNA-Seq | 2.0 GB |
| Cas13d_CYFIP2_gRNA_1_R1_001.fastq.gz | RNA-Seq | 2.7 GB |
| Cas13d_CYFIP2_gRNA_2_R1_001.fastq.gz | RNA-Seq | 3.9 GB |
| Cas13d_NT_gRNA_1_R1_001.fastq.gz | RNA-Seq | 1.3 GB |
| Cas13d_NT_gRNA_2_R1_001.fastq.gz | RNA-Seq | 3.9 GB |
| Cas9_CYFIP2_gRNA_1_R1_001.fastq.gz | RNA-Seq | 4.4 GB |
| Cas9_CYFIP2_gRNA_2_R1_001.fastq.gz | RNA-Seq | 6.2 GB |
| Cas9_NT_gRNA_1_R1_001.fastq.gz | RNA-Seq | 1.2 GB |
| Cas9_NT_gRNA_2_R1_001.fastq.gz | RNA-Seq | 1.3 GB |
| SRR12036520_GSM4623716 | RNA-Seq | 4.2 GB |
| SRR12036521_GSM4623717 | RNA-Seq | 2.7 GB |
| SRR12036522_GSM4623718 | RNA-Seq | 4.3 GB |
| SRR12036523_GSM4623719 | RNA-Seq | 2.0 GB |
| SRR12036524_GSM4623720 | RNA-Seq | 2.7 GB |
| SRR12036525_GSM4623721 | RNA-Seq | 3.9 GB |
| SRR12036526_GSM4623722 | RNA-Seq | 1.3 GB |
| SRR12036527_GSM4623723 | RNA-Seq | 3.9 GB |
| SRR12036528_GSM4623724 | RNA-Seq | 4.4 GB |
| SRR12036529_GSM4623725 | RNA-Seq | 6.2 GB |
| SRR12036530_GSM4623726 | RNA-Seq | 1.2 GB |
| SRR12036531_GSM4623727 | RNA-Seq | 1.3 GB |
| SRR12036532_GSM4623728 | RNA-Seq | 587.0 MB |
| SRR12036533_GSM4623729 | RNA-Seq | 717.0 MB |
| SRR12036534_GSM4623730 | RNA-Seq | 950.0 MB |
| Untransfected_293T_1_R1_001.fastq.gz | RNA-Seq | 587.6 MB |
| Untransfected_293T_2_R1_001.fastq.gz | RNA-Seq | 717.3 MB |
| Untransfected_293T_3_R1_001.fastq.gz | RNA-Seq | 950.3 MB |
Potentially related datasets 1
These accessions were text-mined from the PMC full text. They may be referenced for comparison, cited from other studies, or otherwise mentioned without being primary data for this paper.
Analysis pipelines 1
| Pipeline | Assay | Steps | Dataset |
|---|---|---|---|
| GSE152684 Data Processing geo_data_processing | — | 8 | GSE152684 |
Funding 8 grants
| Grant | Funder |
|---|---|
| S10 OD026929 | NIH HHS (United States) |
| R01 HG004659 | NHGRI NIH HHS (United States) |
| R01 NS103172 | NINDS NIH HHS (United States) |
| T32 GM145427 | NIGMS NIH HHS (United States) |
| K22 NS112678 | NINDS NIH HHS (United States) |
| T32 GM008666 | NIGMS NIH HHS (United States) |
| R01 EY029166 | NEI NIH HHS (United States) |
| F31 NS111859 | NINDS NIH HHS (United States) |