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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
PubMed DOI PMC Full Text

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.

Publication Types

["Journal Article", "Research Support, N.I.H., Extramural"]

Keywords

["ADAR2", "CRISPR", "Cas13", "Cas9", "RNA editing", "RNA targeting Cas9", "spacerless guide RNA", "transcriptome engineering"]

MeSH Terms

["Adenosine Deaminase", "Base Sequence", "CRISPR-Associated Protein 9", "CRISPR-Cas Systems", "Genetic Engineering", "HEK293 Cells", "Humans", "RNA Editing", "RNA, Guide, CRISPR-Cas Systems", "RNA-Binding Proteins", "RNA-Seq", "Transcriptome"]

Funding

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)

Linked Datasets (1)

GSE152684 GSE via ncbi_elink
GEO

Discovery and evaluation of CRISPR/Cas-mediated systems for site-specific RNA editing

Homo sapiens
30 data files
FileTypeSize
Cas13b_CYFIP2_gRNA_1_R1_001.fastq.gz RNA-Seq 4.2 GB
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_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_1_R1_001.fastq.gz RNA-Seq 4.3 GB
Cas13b_NT_gRNA_2_R1_001.fastq.gz RNA-Seq 2.0 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_1_R1_001.fastq.gz RNA-Seq 2.7 GB
Cas13d_CYFIP2_gRNA_2_R1_001.fastq.gz RNA-Seq 3.9 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_1_R1_001.fastq.gz RNA-Seq 1.3 GB
Cas13d_NT_gRNA_2_R1_001.fastq.gz RNA-Seq 3.9 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_1_R1_001.fastq.gz RNA-Seq 4.4 GB
Cas9_CYFIP2_gRNA_2_R1_001.fastq.gz RNA-Seq 6.2 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_1_R1_001.fastq.gz RNA-Seq 1.2 GB
Cas9_NT_gRNA_2_R1_001.fastq.gz RNA-Seq 1.3 GB
Cas9_NT_gRNA_2_R1_001.fastq.gz RNA-Seq 1.3 GB
Untransfected_293T_1_R1_001.fastq.gz RNA-Seq 587.6 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_2_R1_001.fastq.gz RNA-Seq 717.3 MB
Untransfected_293T_3_R1_001.fastq.gz RNA-Seq 950.3 MB
Untransfected_293T_3_R1_001.fastq.gz RNA-Seq 950.3 MB

Analysis Pipelines (1)

GSE152684 Data Processing 8 steps
geo_data_processing GSE152684