I am a lover of all things RNA currently interested in studying sub-cellular localization of mRNA molecules. Using primary oligodendrocytes as a model system, I am studying the mechanisms of mRNA transport in myelin development.
Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics.
2022; 13 (1): 1536
Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop an RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that highly structured "superfolder" mRNAs can be designed to improve both stability and expression with further enhancement through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.
View details for DOI 10.1038/s41467-022-28776-w
View details for PubMedID 35318324
Defining genome-wide CRISPR-Cas genome-editing nuclease activity with GUIDE-seq.
Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of genome-editing nucleases in living cells. GUIDE-seq is based on the principle of efficient integration of an end-protected double-stranded oligodeoxynucleotide tag into sites of nuclease-induced DNA double-stranded breaks, followed by amplification of tag-containing genomic DNA molecules and high-throughput sequencing. Here we describe a detailed GUIDE-seq protocol including cell transfection, library preparation, sequencing and bioinformatic analysis. The entire protocol including cell culture can be completed in 9 d. Once tag-integrated genomic DNA is isolated, library preparation, sequencing and analysis can be performed in 3 d. The result is a genome-wide catalog of off-target sites ranked by nuclease activity as measured by GUIDE-seq read counts. GUIDE-seq is one of the most sensitive cell-based methods for defining genome-wide off-target activity and has been broadly adopted for research and therapeutic use.
View details for DOI 10.1038/s41596-021-00626-x
View details for PubMedID 34773119
mRNA Transport and Local Translation in Glia.
Trends in cell biology
Though mRNA transport and local translation are extensively studied in neurons, emerging evidence supports that these cellular processes are also abundant in non-neuronal glial cells. Here, we explore mechanisms of mRNA transport and local translation in oligodendrocytes, astrocytes, microglia, radial glia, and their functions in development, structure, and intercellular interactions.
View details for DOI 10.1016/j.tcb.2021.03.006
View details for PubMedID 33840591
Anomalous Reverse Transcription through Chemical Modifications in Polyadenosine Stretches.
Thermostable reverse transcriptases are workhorse enzymes underlying nearly all modern techniques for RNA structure mapping and for the transcriptome-wide discovery of RNA chemical modifications. Despite their wide use, these enzymes' behaviors at chemical modified nucleotides remain poorly understood. Wellington-Oguri et al. recently reported an apparent loss of chemical modification within putatively unstructured polyadenosine stretches modified by dimethyl sulfate or 2' hydroxyl acylation, as probed by reverse transcription. Here, reanalysis of these and other publicly available data, capillary electrophoresis experiments on chemically modified RNAs, and nuclear magnetic resonance spectroscopy on (A)12 and variants show that this effect is unlikely to arise from an unusual structure of polyadenosine. Instead, tests of different reverse transcriptases on chemically modified RNAs and molecules synthesized with single 1-methyladenosines implicate a previously uncharacterized reverse transcriptase behavior: near-quantitative bypass through chemical modifications within polyadenosine stretches. All tested natural and engineered reverse transcriptases (MMLV; SuperScript II, III, and IV; TGIRT-III; and MarathonRT) exhibit this anomalous bypass behavior. Accurate DMS-guided structure modeling of the polyadenylated HIV-1 3' untranslated region requires taking into account this anomaly. Our results suggest that poly(rA-dT) hybrid duplexes can trigger an unexpectedly effective reverse transcriptase bypass and that chemical modifications in mRNA poly(A) tails may be generally undercounted.
View details for DOI 10.1021/acs.biochem.0c00020
View details for PubMedID 32407625
Structural Determinants of MRNA Transport Specificity in Oligodendrocytes
CELL PRESS. 2020: 67A
View details for Web of Science ID 000513023200334
Accelerated cryo-EM-guided determination of three-dimensional RNA-only structures.
2020; 17 (7): 699–707
The discovery and design of biologically important RNA molecules is outpacing three-dimensional structural characterization. Here, we demonstrate that cryo-electron microscopy can routinely resolve maps of RNA-only systems and that these maps enable subnanometer-resolution coordinate estimation when complemented with multidimensional chemical mapping and Rosetta DRRAFTER computational modeling. This hybrid 'Ribosolve' pipeline detects and falsifies homologies and conformational rearrangements in 11 previously unknown 119- to 338-nucleotide protein-free RNA structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length Vibrio cholerae and Fusobacterium nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and the computer-designed ATP-TTR-3 aptamer with and without AMP. Simulation benchmarks, blind challenges, compensatory mutagenesis, cross-RNA homologies and internal controls demonstrate that Ribosolve can accurately resolve the global architectures of RNA molecules but does not resolve atomic details. These tests offer guidelines for making inferences in future RNA structural studies with similarly accelerated throughput.
View details for DOI 10.1038/s41592-020-0878-9
View details for PubMedID 32616928
Transcription factors, coregulators, and epigenetic marks are linearly correlated and highly redundant
2017; 12 (12): e0186324
The DNA microstates that regulate transcription include sequence-specific transcription factors (TFs), coregulatory complexes, nucleosomes, histone modifications, DNA methylation, and parts of the three-dimensional architecture of genomes, which could create an enormous combinatorial complexity across the genome. However, many proteins and epigenetic marks are known to colocalize, suggesting that the information content encoded in these marks can be compressed. It has so far proved difficult to understand this compression in a systematic and quantitative manner. Here, we show that simple linear models can reliably predict the data generated by the ENCODE and Roadmap Epigenomics consortia. Further, we demonstrate that a small number of marks can predict all other marks with high average correlation across the genome, systematically revealing the substantial information compression that is present in different cell lines. We find that the linear models for activating marks are typically cell line-independent, while those for silencing marks are predominantly cell line-specific. Of particular note, a nuclear receptor corepressor, transducin beta-like 1 X-linked receptor 1 (TBLR1), was highly predictive of other marks in two hematopoietic cell lines. The methodology presented here shows how the potentially vast complexity of TFs, coregulators, and epigenetic marks at eukaryotic genes is highly redundant and that the information present can be compressed onto a much smaller subset of marks. These findings could be used to efficiently characterize cell lines and tissues based on a small number of diagnostic marks and suggest how the DNA microstates, which regulate the expression of individual genes, can be specified.
View details for DOI 10.1371/journal.pone.0186324
View details for Web of Science ID 000417337800003
View details for PubMedID 29216191
View details for PubMedCentralID PMC5720766
CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR Cas9 nuclease off-targets
2017; 14 (6): 607-+
Sensitive detection of off-target effects is important for translating CRISPR-Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer the potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR-Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequences. Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific single-nucleotide polymorphisms, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of CRISPR-Cas9.
View details for DOI 10.1038/NMETH.4278
View details for Web of Science ID 000402291800024
View details for PubMedID 28459458
View details for PubMedCentralID PMC5924695
- Open-source guideseq software for analysis of GUIDE-seq data NATURE BIOTECHNOLOGY 2016; 34 (5): 483
Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition
2015; 33 (12): 1293-+
CRISPR-Cas9 nucleases target specific DNA sequences using a guide RNA but also require recognition of a protospacer adjacent motif (PAM) by the Cas9 protein. Although longer PAMs can potentially improve the specificity of genome editing, they limit the range of sequences that Cas9 orthologs can target. One potential strategy to relieve this restriction is to relax the PAM recognition specificity of Cas9. Here we used molecular evolution to modify the NNGRRT PAM of Staphylococcus aureus Cas9 (SaCas9). One variant we identified, referred to as KKH SaCas9, showed robust genome editing activities at endogenous human target sites with NNNRRT PAMs, thereby increasing SaCas9 targeting range by two- to fourfold. Using GUIDE-seq, we show that wild-type and KKH SaCas9 induce comparable numbers of off-target effects in human cells. Our strategy for evolving PAM specificity does not require structural information and therefore should be applicable to a wide range of Cas9 orthologs.
View details for DOI 10.1038/nbt.3404
View details for Web of Science ID 000366387700022
View details for PubMedID 26524662
View details for PubMedCentralID PMC4689141
Engineered CRISPR-Cas9 nucleases with altered PAM specificities
2015; 523 (7561): 481-U249
Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.
View details for DOI 10.1038/nature14592
View details for Web of Science ID 000358378900041
View details for PubMedID 26098369
View details for PubMedCentralID PMC4540238
Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing
HUMAN GENE THERAPY
2015; 26 (7): 425-431
Monomeric clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) nucleases have been widely adopted for simple and robust targeted genome editing but also have the potential to induce high-frequency off-target mutations. In principle, two orthogonal strategies for reducing off-target cleavage, truncated guide RNAs (tru-gRNAs) and dimerization-dependent RNA-guided FokI-dCas9 nucleases (RFNs), could be combined as tru-RFNs to further improve genome editing specificity. Here we identify a robust tru-RFN architecture that shows high activity in human cancer cell lines and embryonic stem cells. Additionally, we demonstrate that tru-gRNAs reduce the undesirable mutagenic effects of monomeric FokI-dCas9. Tru-RFNs combine the advantages of two orthogonal strategies for improving the specificity of CRISPR-Cas nucleases and therefore provide a highly specific platform for performing genome editing.
View details for DOI 10.1089/hum.2015.084
View details for Web of Science ID 000362083000005
View details for PubMedID 26068112
View details for PubMedCentralID PMC4509490
- Defining Genome-Wide Off-Target Cleavage Profiles of CRISPR-Cas RNA-Guided Nucleases Using GUIDE-Seq NATURE PUBLISHING GROUP. 2015: S274
- Engineered Cas9 Variants with Novel PAM Specificities Expand the Targeting Range of CRISPR/Cas Nucleases NATURE PUBLISHING GROUP. 2015: S26
Replacing Uridine with 2-Thiouridine Enhances the Rate and Fidelity of Nonenzymatic RNA Primer Extension
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2015; 137 (7): 2769-2775
The nonenzymatic replication of RNA oligonucleotides is thought to have played a key role in the origin of life prior to the evolution of ribozyme-catalyzed RNA replication. Although the copying of oligo-C templates by 2-methylimidazole-activated G monomers can be quite efficient, the copying of mixed sequence templates, especially those containing A and U, is particularly slow and error-prone. The greater thermodynamic stability of the 2-thio-U(s(2)U):A base pair, relative to the canonical U:A base pair, suggests that replacing U with s(2)U might enhance the rate and fidelity of the nonenzymatic copying of RNA templates. Here we report that this single atom substitution in the activated monomer improves both the kinetics and the fidelity of nonenzymatic primer extension on mixed-sequence RNA templates. In addition, the mean lengths of primer extension products obtained with s(2)U is greater than those obtained with U, augmenting the potential for nonenzymatic replication of heritable function-rich sequences. We suggest that noncanonical nucleotides such as s(2)U may have played a role during the infancy of the RNA world by facilitating the nonenzymatic replication of genomic RNA oligonucleotides.
View details for DOI 10.1021/jacs.5b00445
View details for Web of Science ID 000350192700050
View details for PubMedID 25654265
View details for PubMedCentralID PMC4985000
GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases
2015; 33 (2): 187-197
CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide, off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint 'hotspots'. Finally, GUIDE-seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced, off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases before clinical use.
View details for DOI 10.1038/nbt.3117
View details for Web of Science ID 000349198800025
View details for PubMedID 25513782
View details for PubMedCentralID PMC4320685