scholarly journals CRISPRO Identifies Functional Protein Coding Sequences Based on Genome Editing Dense Mutagenesis

2018 ◽  
Author(s):  
Vivien A. C. Schoonenberg ◽  
Mitchel A. Cole ◽  
Qiuming Yao ◽  
Claudio Macias-Treviño ◽  
Falak Sher ◽  
...  
Keyword(s):  
Machine Learning ◽  
Computational Pipeline ◽  
Functional Protein ◽  
Protein Coding ◽  
Coding Sequences ◽  
Guide Rna ◽  
Guide Rnas ◽  
Functional Scores ◽  
Parallel Evaluation

AbstractCRISPR/Cas9 pooled screening permits parallel evaluation of comprehensive guide RNA libraries to systematically perturb protein coding sequences in situ and correlate with functional readouts. For the analysis and visualization of the resulting datasets we have developed CRISPRO, a computational pipeline that maps functional scores associated with guide RNAs to genome, transcript, and protein coordinates and structure. No available tool has similar functionality. The ensuing genotype-phenotype linear and 3D maps raise hypotheses about structure-function relationships at discrete protein regions. Machine learning based on CRISPRO features improves prediction of guide RNA efficacy. The CRISPRO tool is freely available at gitlab.com/bauerlab/crispro.

scholarly journals A modular cloning toolkit for genome editing in plants

2019 ◽  
Author(s):  
Florian Hahn ◽  
Andrey Korolev ◽  
Laura Sanjurjo Loures ◽  
Vladimir Nekrasov
Keyword(s):  
Genome Editing ◽  
Higher Order ◽  
Plant Science ◽  
Golden Gate ◽  
Coding Sequences ◽  
Guide Rna ◽  
Guide Rnas ◽  
Science Community ◽  
Modular Cloning ◽  
Multiple Expression

AbstractBackgroundCRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants. Applying CRISPR/Cas often requires delivering multiple expression units into plant and hence there is a need for a quick and easy cloning procedure. The modular cloning (MoClo), based on the Golden Gate (GG) method, has enabled development of cloning systems with standardised genetic parts, e.g. promoters, coding sequences or terminators, that can be easily interchanged and assembled into expression units, which in their own turn can be further assembled into higher order multigene constructs.ResultsHere we present an expanded cloning toolkit that contains ninety-nine modules encoding a variety of CRISPR/Cas-based nucleases and their corresponding guide RNA backbones. Among other components, the toolkit includes a number of promoters that allow expression of CRISPR/Cas nucleases (or any other coding sequences) and their guide RNAs in monocots and dicots. As part of the toolkit, we present a set of modules that enable quick and facile assembly of tRNA-sgRNA polycistronic units without a PCR step involved. We also demonstrate that our tRNA-sgRNA system is functional in wheat protoplasts.ConclusionsWe believe the presented CRISPR/Cas toolkit is a great resource that will contribute towards wider adoption of the CRISPR/Cas genome editing technology and modular cloning by researchers across the plant science community.

scholarly journals Cellular thermal shift analysis for interrogation of CRISPR-assisted proteomic changes

BioTechniques ◽  
2020 ◽  
Vol 68 (4) ◽  
pp. 180-184 ◽  
Author(s):  
Nam-Gu Her ◽  
Ivan Babic ◽  
Venkata M Yenugonda ◽  
Santosh Kesari ◽  
Elmar Nurmemmedov
Keyword(s):  
Protein Complexes ◽  
Physiological State ◽  
Protein Coding ◽  
Coding Sequences ◽  
Missing Link ◽  
Disease States ◽  
Shift Analysis ◽  
Versatile Tool ◽  
Thermal Shift

CRISPR–Cas9 has proven to be a versatile tool for the discovery of essential genetic elements involved in various disease states. CRISPR-assisted dense mutagenesis focused on therapeutically challenging protein complexes allows us to systematically perturb protein-coding sequences in situ and correlate them with functional readouts. Such perturbations can mimic targeting by therapeutics and serve as a foundation for the discovery of highly specific modulators. However, translation of such genomics data has been challenging due to the missing link for proteomics under the physiological state of the cell. We present a method based on cellular thermal shift assays to easily interrogate proteomic shifts generated by CRISPR-assisted dense mutagenesis, as well as a case focused on NuRD epigenetic complex.

scholarly journals Transcriptome-wide Cas13 guide RNA design for model organisms and viral RNA pathogens

2020 ◽  
Author(s):  
Xinyi Guo ◽  
Hans-Hermann Wessels ◽  
Alejandro Méndez-Mancilla ◽  
Daniel Haro ◽  
Neville E. Sanjana
Keyword(s):  
Rna Virus ◽  
H1n1 Influenza ◽  
Model Organisms ◽  
Messenger Rnas ◽  
Protein Coding ◽  
Guide Rna ◽  
Knock Down ◽  
Guide Rnas ◽  
Rna Targeting ◽  
Rna Design

AbstractCRISPR-Cas13 mediates robust transcript knockdown in human cells through direct RNA targeting. Compared to DNA-targeting CRISPR enzymes like Cas9, RNA targeting by Cas13 is transcript- and strand-specific: It can distinguish and specifically knock-down processed transcripts, alternatively spliced isoforms and overlapping genes, all of which frequently serve different functions. Previously, we identified optimal design rules for RfxCas13d guide RNAs (gRNAs), and developed a computational model to predict gRNA efficacy for all human protein-coding genes. However, there is a growing interest to target other types of transcripts, such as noncoding RNAs (ncRNAs) or viral RNAs, and to target transcripts in other commonly-used organisms. Here, we predicted relative Cas13-driven knock-down for gRNAs targeting messenger RNAs and ncRNAs in six model organisms (human, mouse, zebrafish, fly, nematode and flowering plants) and four abundant RNA virus families (SARS-CoV-2, HIV-1, H1N1 influenza and MERS). To allow for more flexible gRNA efficacy prediction, we also developed a web-based application to predict optimal gRNAs for any RNA target entered by the user. Given the lack of Cas13 guide design tools, we anticipate this resource will facilitate CRISPR-Cas13 RNA targeting in common model organisms, emerging viral threats to human health, and novel RNA targets.

scholarly journals CRISPRO: identification of functional protein coding sequences based on genome editing dense mutagenesis

2018 ◽  
Vol 19 (1) ◽  
Author(s):  
Vivien A. C. Schoonenberg ◽  
Mitchel A. Cole ◽  
Qiuming Yao ◽  
Claudio Macias-Treviño ◽  
Falak Sher ◽  
...  
Keyword(s):  
Genome Editing ◽  
Functional Protein ◽  
Protein Coding ◽  
Coding Sequences

scholarly journals Machine learning based CRISPR gRNA design for therapeutic exon skipping

2021 ◽  
Vol 17 (1) ◽  
pp. e1008605
Author(s):  
Wilson Louie ◽  
Max W. Shen ◽  
Zakir Tahiry ◽  
Sophia Zhang ◽  
Daniel Worstell ◽  
...  
Keyword(s):  
Machine Learning ◽  
Stem Cells ◽  
Gene Function ◽  
Genetic Disease ◽  
Genetic Disorders ◽  
Embryonic Stem ◽  
Exon Skipping ◽  
Therapeutic Modality ◽  
Guide Rna ◽  
Guide Rnas

Restoring gene function by the induced skipping of deleterious exons has been shown to be effective for treating genetic disorders. However, many of the clinically successful therapies for exon skipping are transient oligonucleotide-based treatments that require frequent dosing. CRISPR-Cas9 based genome editing that causes exon skipping is a promising therapeutic modality that may offer permanent alleviation of genetic disease. We show that machine learning can select Cas9 guide RNAs that disrupt splice acceptors and cause the skipping of targeted exons. We experimentally measured the exon skipping frequencies of a diverse genome-integrated library of 791 splice sequences targeted by 1,063 guide RNAs in mouse embryonic stem cells. We found that our method, SkipGuide, is able to identify effective guide RNAs with a precision of 0.68 (50% threshold predicted exon skipping frequency) and 0.93 (70% threshold predicted exon skipping frequency). We anticipate that SkipGuide will be useful for selecting guide RNA candidates for evaluation of CRISPR-Cas9-mediated exon skipping therapy.

scholarly journals Human Box H/ACA Pseudouridylation Guide RNA Machinery

2004 ◽  
Vol 24 (13) ◽  
pp. 5797-5807 ◽  
Author(s):  
Arnold M. Kiss ◽  
Beáta E. Jády ◽  
Edouard Bertrand ◽  
Tamás Kiss
Keyword(s):  
Protein Product ◽  
Protein Coding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Small Nuclear Rnas ◽  
Spliceosomal Rnas ◽  
Coding Capacity ◽  
Rna Genes ◽  
Specific Synthesis

ABSTRACT Pseudouridine, the most abundant modified nucleoside in RNA, is synthesized by posttranscriptional isomerization of uridines. In eukaryotic RNAs, site-specific synthesis of pseudouridines is directed primarily by box H/ACA guide RNAs. In this study, we have identified 61 novel putative pseudouridylation guide RNAs by construction and characterization of a cDNA library of human box H/ACA RNAs. The majority of the new box H/ACA RNAs are predicted to direct pseudouridine synthesis in rRNAs and spliceosomal small nuclear RNAs. We can attribute RNA-directed modification to 79 of the 97 pseudouridylation sites present in the human 18S, 5.8S, and 28S rRNAs and to 11 of the 21 pseudouridines reported for the U1, U2, U4, U5, and U6 spliceosomal RNAs. We have also identified 12 novel box H/ACA RNAs which lack apparent target pseudouridines in rRNAs and small nuclear RNAs. These putative guide RNAs likely function in the pseudouridylation of some other types of cellular RNAs, suggesting that RNA-guided pseudouridylation is more general than assumed before. The genomic organization of the new box H/ACA RNA genes indicates that in human cells, all box H/ACA pseudouridylation guide RNAs are processed from introns of pre-mRNA transcripts which either encode a protein product or lack protein-coding capacity.

scholarly journals De novo gene evolution: How do we transition from non-coding to coding?

2017 ◽  
Author(s):  
Jorge Ruiz-Orera ◽  
José Luis Villanueva-Cañas ◽  
William Blevins ◽  
M.Mar Albà
Keyword(s):  
De Novo ◽  
Gene Evolution ◽  
Neutral Evolution ◽  
Functional Protein ◽  
Protein Coding ◽  
Coding Sequences ◽  
Sequence Composition ◽  
Protein Coding Genes ◽  
Small Proteins ◽  
De Novo Gene

Recent years have witnessed the discovery of protein–coding genes which appear to have evolved de novo from previously non-coding sequences. This has changed the long-standing view that coding sequences can only evolve from other coding sequences. However, there are still many open questions regarding how new protein-coding sequences can arise from non-genic DNA. Two prerequisites for the birth of a new functional protein-coding gene are that the corresponding DNA fragment is transcribed and that it is also translated. Transcription is known to be pervasive in the genome, producing a large number of transcripts that do not correspond to conserved protein-coding genes, and which are usually annotated as long non-coding RNAs (lncRNA). Recently, sequencing of ribosome protected fragments (Ribo-Seq) has provided evidence that many of these transcripts actually translate small proteins. We have used mouse non-synonymous and synonymous variation data to estimate the strength of purifying selection acting on the translated open reading frames (ORFs). Whereas a subset of the lncRNAs are likely to actually be true protein-coding genes (and thus previously misclassified), the bulk of lncRNAs code for proteins which show variation patterns consistent with neutral evolution. We also show that the ORFs that have a more favorable, coding-like, sequence composition are more likely to be translated than other ORFs in lncRNAs. This study provides strong evidence that there is a large and ever-changing reservoir of lowly abundant proteins; some of these peptides may become useful and act as seeds for de novo gene evolution.

scholarly journals Principles for rational Cas13d guide design

2019 ◽  
Author(s):  
Hans-Hermann Wessels ◽  
Alejandro Méndez-Mancilla ◽  
Xinyi Guo ◽  
Mateusz Legut ◽  
Zharko Daniloski ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Perfect Match ◽  
Nuclease Activity ◽  
Target Site ◽  
Surface Proteins ◽  
Protein Coding ◽  
Guide Rna ◽  
Knock Down ◽  
Guide Rnas ◽  
Green Fluorescent

AbstractType VI CRISPR enzymes have recently been identified as programmable RNA-guided, RNA-targeting Cas proteins with nuclease activity that allow for specific and robust target gene knock-down without altering the genome. However, we currently lack information about optimal Cas13 guide RNA designs for high target RNA knock-down efficacy. To close this gap, we conducted four massively-parallel Cas13 screens targeting the mRNA of a destabilized green fluorescent protein (GFP) transgene and CD46, CD55 and CD71 cell surface proteins in human cells. In total, we measured the activity of 24,460 guide RNA including 6,469 perfect match guide RNAs and a diverse set of guide RNA variants and permutations with mismatches relative to the target sequences.We find that guide RNAs show high diversity in knock-down efficiency driven by crRNA-specific features as well as target site context. Moreover, while single mismatches generally reduce knock-down to a modest degree, we identify a critical region spanning spacer nucleotides 15 – 21 that is largely intolerant to target site mismatches. We developed a computational model to identify guide RNAs with high knock-down efficacy. We confirmed the model’s generalizability across a large number of endogenous target mRNAs and show that Cas13 can be used in forward genetic pooled CRISPR-screens to identify essential genes. Using this model, we provide a resource of optimized Cas13 guide RNAs to target all protein-coding transcripts in the human genome, enabling transcriptome-wide forward genetic screens.

scholarly journals How do we transition from non-coding to coding?

2017 ◽  
Author(s):  
Jorge Ruiz-Orera ◽  
José Luis Villanueva-Cañas ◽  
William Blevins ◽  
M.Mar Albà
Keyword(s):  
De Novo ◽  
Gene Evolution ◽  
Purifying Selection ◽  
Neutral Evolution ◽  
Functional Protein ◽  
Protein Coding ◽  
Coding Sequences ◽  
Sequence Composition ◽  
Protein Coding Genes ◽  
Small Proteins

Recent years have witnessed the discovery of protein–coding genes which appear to have evolved de novo from previously non-coding sequences. This has changed the long-standing view that coding sequences can only evolve from other coding sequences. However, there are still many open questions regarding how new protein-coding sequences can arise from non-genic DNA. Two prerequisites for the birth of a new functional protein-coding gene are that the corresponding DNA fragment is transcribed and that it is also translated. Transcription is known to be pervasive in the genome, producing a large number of transcripts that do not correspond to conserved protein-coding genes, and which are usually annotated as long non-coding RNAs (lncRNA). Recently, sequencing of ribosome protected fragments (Ribo-Seq) has provided evidence that many of these transcripts actually translate small proteins. We have used mouse non-synonymous and synonymous variation data to estimate the strength of purifying selection acting on the translated open reading frames (ORFs). Whereas a subset of the lncRNAs are likely to actually be true protein-coding genes (and thus previously misclassified), the bulk of lncRNAs code for proteins which show variation patterns consistent with neutral evolution. We also show that the ORFs that have a more favorable, coding-like, sequence composition are more likely to be translated than other ORFs in lncRNAs. This study provides strong evidence that there is a large and ever-changing reservoir of lowly abundant proteins; some of these peptides may become useful and act as seeds for de novo gene evolution.

Genome organisation in the murine sperm nucleus

Zygote ◽  
1995 ◽  
Vol 3 (2) ◽  
pp. 123-131 ◽  
Author(s):  
Carol Jennings ◽  
Don Powell
Keyword(s):  
Repetitive Dna ◽  
Ribosomal Dna ◽  
Dna Sequences ◽  
Sperm Nucleus ◽  
Genome Organisation ◽  
Telomeric Dna ◽  
Protein Coding ◽  
Coding Sequences ◽  
Restricted Domains

SummaryThe organisation of DNA sequences in the murine sperm nucleus was studied using in situ hybridisation of biotinylated DNA probes. The efficiency of this reaction was assessed using a dispersed repetitive DNA probe. Telomeric DNA was distributed around the nucleus. Centromeric and ribosomal DNA sequences occupied restricted domains in the sperm nucleus. DNA sequences for a transgene and a cluster of homeogenes occupied different, and rather less defined, domains. Together these results imply that both repetitive and protein-coding sequences are arranged in the nucleus in an ordered fashion.

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