Precise transcript targeting by CRISPR-Csm complexes. Nature biotechnology Robust and precise transcript targeting in mammalian cells remains a difficult challenge using existing approaches due to inefficiency, imprecision and subcellular compartmentalization. Here we show that the clustered regularly interspaced short palindromic repeats (CRISPR)-Csm complex, a multiprotein effector from type III CRISPR immune systems in prokaryotes, provides surgical RNA ablation of both nuclear and cytoplasmic transcripts. As part of the most widely occurring CRISPR adaptive immune pathway, CRISPR-Csm uses a programmable RNA-guided mechanism to find and degrade target RNA molecules without inducing indiscriminate trans-cleavage of cellular RNAs, giving it an important advantage over the CRISPR-Cas13 family of enzymes. Using single-vector delivery of the Streptococcus thermophilus Csm complex, we observe high-efficiency RNA knockdown (90-99%) and minimal off-target effects in human cells, outperforming existing technologies including short hairpin RNA- and Cas13-mediated knockdown. We also find that catalytically inactivated Csm achieves specific and durable RNA binding, a property we harness for live-cell RNA imaging. These results establish the feasibility and efficacy of multiprotein CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes. 10.1038/s41587-022-01649-9

Genome-wide investigation and functional analysis of RNA editing sites in wheat. PloS one Wheat is an important cereal and half of the world population consumed it. Wheat faces environmental stresses and different techniques (CRISPR, gene silencing, GWAS, etc.) were used to enhance its production but RNA editing (RESs) is not fully explored in wheat. RNA editing has a special role in controlling environmental stresses. The genome-wide identification and functional characterization of RESs in different types of wheat genotypes was done. We employed six wheat genotypes by RNA-seq analyses to achieve RESs. The findings revealed that RNA editing events occurred on all chromosomes equally. RNA editing sites were distributed randomly and 10-12 types of RESs were detected in wheat genotypes. Higher number of RESs were detected in drought-tolerant genotypes. A-to-I RNA editing (2952, 2977, 1916, 2576, 3422, and 3459) sites were also identified in six wheat genotypes. Most of the genes were found to be engaged in molecular processes after a Gene Ontology analysis. PPR (pentatricopeptide repeat), OZ1 (organelle zinc-finger), and MORF/RIP gene expression levels in wheat were also examined. Normal growth conditions diverge gene expression of these three different gene families, implying that normal growth conditions for various genotypes can modify RNA editing events and have an impact on gene expression levels. While the expression of PPR genes was not change. We used Variant Effect Predictor (VEP) to annotate RNA editing sites, and Local White had the highest RESs in the CDS region of the protein. These findings will be useful for prediction of RESs in other crops and will be helpful in drought tolerance development in wheat. 10.1371/journal.pone.0265270

Rational Design of RNA Editing Guide Strands: Cytidine Analogs at the Orphan Position. Journal of the American Chemical Society Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in double stranded RNA. Human ADARs can be directed to predetermined target sites in the transcriptome by complementary guide strands, allowing for the correction of disease-causing mutations at the RNA level. Here we use structural information available for ADAR2-RNA complexes to guide the design of nucleoside analogs for the position in the guide strand that contacts a conserved glutamic acid residue in ADARs (E488 in human ADAR2), which flips the adenosine into the ADAR active site for deamination. Mutating this residue to glutamine (E488Q) results in higher activity because of the hydrogen bond donating ability of Q488 to N3 of the orphan cytidine on the guide strand. We describe the evaluation of cytidine analogs for this position that stabilize an activated conformation of the enzyme-RNA complex and increase catalytic rate for deamination by the wild-type enzyme. A new crystal structure of ADAR2 bound to duplex RNA bearing a cytidine analog revealed a close contact between E488, stabilized by an additional hydrogen bond and altered charge distribution when compared to cytidine. In human cells and mouse primary liver fibroblasts, this single nucleotide modification increased directed editing yields when compared to an otherwise identical guide oligonucleotide. Our results show that modification of the guide RNA can mimic the effect of hyperactive mutants and advance the approach of recruiting endogenous ADARs for site-directed RNA editing. 10.1021/jacs.0c13319

RNA editing contributes to epitranscriptome diversity in chronic lymphocytic leukemia. Gassner Franz J,Zaborsky Nadja,Buchumenski Ilana,Levanon Erez Y,Gatterbauer Matthias,Schubert Maria,Rauscher Stefanie,Hebenstreit Daniel,Nadeu Ferran,Campo Elias,Egle Alexander,Greil Richard,Geisberger Roland Leukemia RNA editing-primarily conversion of adenosine to inosine (A > I)-is a widespread posttranscriptional mechanism, mediated by Adenosine Deaminases acting on RNA (ADAR) enzymes to alter the RNA sequence of primary transcripts. Hence, in addition to somatic mutations and alternative RNA splicing, RNA editing can be a further source for recoding events. Although RNA editing has been detected in many solid cancers and normal tissue, RNA editing in chronic lymphocytic leukemia (CLL) has not been addressed so far. We determined global RNA editing and recurrent, recoding RNA editing events from matched RNA-sequencing and whole exome sequencing data in CLL samples from 45 untreated patients. RNA editing was verified in a validation cohort of 98 CLL patients and revealed substantially altered RNA editing profiles in CLL compared with normal B cells. We further found that RNA editing patterns were prognostically relevant. Finally, we showed that ADAR knockout decreased steady state viability of MEC1 cells and made them more susceptible to treatment with fludarabine and ibrutinib in vitro. We propose that RNA editing contributes to the pathophysiology of CLL and targeting the RNA editing machinery could be a future strategy to maximize treatment efficacy. 10.1038/s41375-020-0995-6