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 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 One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Joseph R. Owen ◽  
Sadie L. Hennig ◽  
Bret R. McNabb ◽  
Tamer A. Mansour ◽  
Justin M. Smith ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Bos Taurus ◽  
Single Copy ◽  
Blastocyst Stage ◽  
Target Site ◽  
End Joining ◽  
Bull Calf ◽  
Dividing Cells ◽  
Green Fluorescent ◽  
Donor Template

Abstract Background The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells. Results By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template. Conclusion The allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template. This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

scholarly journals Supramolecular nanosubstrate–mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies

2020 ◽  
Vol 6 (43) ◽  
pp. eabb7107
Author(s):  
Peng Yang ◽  
Shih-Jie Chou ◽  
Jindian Li ◽  
Wenqiao Hui ◽  
Wenfei Liu ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Integration Site ◽  
Genetic Diseases ◽  
Proliferative Potential ◽  
Adeno Associated Virus ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Green Fluorescent ◽  
Virus Integration

Leveraging the endogenous homology-directed repair (HDR) pathway, the CRISPR-Cas9 gene-editing system can be applied to knock in a therapeutic gene at a designated site in the genome, offering a general therapeutic solution for treating genetic diseases such as hemoglobinopathies. Here, a combined supramolecular nanoparticle (SMNP)/supramolecular nanosubstrate–mediated delivery (SNSMD) strategy is used to facilitate CRISPR-Cas9 knockin of the hemoglobin beta (HBB) gene into the adeno-associated virus integration site 1 (AAVS1) safe-harbor site of an engineered K562 3.21 cell line harboring the sickle cell disease mutation. Through stepwise treatments of the two SMNP vectors encapsulating a Cas9•single-guide RNA (sgRNA) complex and an HBB/green fluorescent protein (GFP)–encoding plasmid, CRISPR-Cas9 knockin was successfully achieved via HDR. Last, the HBB/GFP-knockin K562 3.21 cells were introduced into mice via intraperitoneal injection to show their in vivo proliferative potential. This proof-of-concept demonstration paves the way for general gene therapeutic solutions for treating hemoglobinopathies.

scholarly journals Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii

2014 ◽  
Vol 13 (11) ◽  
pp. 1465-1469 ◽  
Author(s):  
Wenzhi Jiang ◽  
Andrew J. Brueggeman ◽  
Kempton M. Horken ◽  
Thomas M. Plucinak ◽  
Donald P. Weeks
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Transient Expression ◽  
Gene Knockout ◽  
Nuclease Activity ◽  
Gene Replacement ◽  
Target Site ◽  
Gene Encoding ◽  
Content Type ◽  
Guide Rna ◽  
Eukaryotic Organisms

ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii , failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii , we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10 9 cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii . The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing.

scholarly journals Cell Surface Organization of Stress-inducible Proteins ULBP and MICA That Stimulate Human NK Cells and T Cells via NKG2D

2004 ◽  
Vol 199 (7) ◽  
pp. 1005-1010 ◽  
Author(s):  
Konstantina Eleme ◽  
Sabrina B. Taner ◽  
Björn Önfelt ◽  
Lucy M. Collinson ◽  
Fiona E. McCann ◽  
...  
Keyword(s):  
Cell Surface ◽  
Nk Cells ◽  
Lipid Rafts ◽  
Mhc Class I ◽  
Fluorescent Protein ◽  
Nk Cell ◽  
Surface Proteins ◽  
Class I ◽  
Immune Synapse ◽  
Green Fluorescent

Cell surface proteins major histocompatibility complex (MHC) class I–related chain A (MICA) and UL16-binding proteins (ULBP) 1, 2, and 3 are up-regulated upon infection or tumor transformation and can activate human natural killer (NK) cells. Patches of cross-linked raft resident ganglioside GM1 colocalized with ULBP1, 2, 3, or MICA, but not CD45. Thus, ULBPs and MICA are expressed in lipid rafts at the cell surface. Western blotting revealed that glycosylphosphatidylinositol (GPI)-anchored ULBP3 but not transmembrane MICA, MHC class I protein, or transferrin receptor, accumulated in detergent-resistant membranes containing GM1. Thus, MICA may have a weaker association with lipid rafts than ULBP3, yet both proteins accumulate at an activating human NK cell immune synapse. Target cell lipid rafts marked by green fluorescent protein–tagged GPI also accumulate with ULBP3 at some synapses. Electron microscopy reveals constitutive clusters of ULBP at the cell surface. Regarding a specific molecular basis for the organization of these proteins, ULBP1, 2, and 3 and MICA are lipid modified. ULBP1, 2, and 3 are GPI anchored, and we demonstrate here that MICA is S-acylated. Finally, expression of a truncated form of MICA that lacks the putative site for S-acylation and the cytoplasmic tail can be expressed at the cell surface, but is unable to activate NK cells.

scholarly journals Engineered Microbes to Sense and Respond to Enterotoxigenic Escherichia coli

2018 ◽  
Author(s):  
A. Bete ◽  
J. Carter ◽  
C. Davis ◽  
J. Dong ◽  
M. Herrmann ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Genetically Engineered ◽  
Surface Proteins ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Autoinducer 2 ◽  
Binding Domains ◽  
Sense And Respond ◽  
Green Fluorescent

AbstractEvery year, Enterotoxigenic Escherichia coli (ETEC), the most common form of traveler’s diarrhea, affects thousands of military personnel deployed overseas. The goal of this research was to engineer non-pathogenic E. coli to sense ETEC, respond to its presence, and package the non-pathogenic E. coli in a cellulose matrix to enable environmental detection of ETEC. Two plasmids were created: ‘sense-respond’; and ‘packaging’. The sense-respond plasmid detected autoinducer 2 (AI-2), a quorum sensing molecule created by most ETEC strains, by expressing LsrR which switches on the Lsr promoter. Activation of the Lsr promoter expresses superfolder green fluorescent protein (sfGFP), indicating the presence of ETEC. The packaging plasmid expresses a fusion protein consisting of curli fibers and cellulose binding domains. These modified surface proteins permit the bacteria to bind to cellulose, encapsulating the sense-response module. This genetically engineered machine could be deployed in both the internal and external environment to detect ETEC.

scholarly journals Effective RNA Knockdown Using CRISPR-Cas13a and Molecular Targeting of the EML4-ALK Transcript in H3122 Lung Cancer Cells

2020 ◽  
Vol 21 (23) ◽  
pp. 8904
Author(s):  
Saifullah ◽  
Matomo Sakari ◽  
Takeshi Suzuki ◽  
Seiji Yano ◽  
Toshifumi Tsukahara
Keyword(s):  
Lung Cancer ◽  
Cancer Cells ◽  
Firefly Luciferase ◽  
Lung Cancer Cells ◽  
Guide Rna ◽  
Knock Down ◽  
Guide Rnas ◽  
Rnai Technology ◽  
Low Efficiency ◽  
The Stability

RNAi technology has significant potential as a future therapeutic and could theoretically be used to knock down disease-specific RNAs. However, due to frequent off-target effects, low efficiency, and limited accessibility of nuclear transcripts, the clinical application of the technology remains challenging. In this study, we first assessed the stability of Cas13a mRNA and guide RNA. Next, we titrated Cas13a and guide RNA vectors to achieve effective knockdown of firefly luciferase (FLuc) RNA, used as a target transcript. The interference specificity of Cas13a on guide RNA design was next explored. Subsequently, we targeted the EML4-ALK v1 transcript in H3122 lung cancer cells. As determined by FLuc assay, Cas13a exhibited activity only toward the orientation of the crRNA–guide RNA complex residing at the 5′ of the crRNA. The activity of Cas13a was maximal for guide RNAs 24–30 bp in length, with relatively low mismatch tolerance. After knockdown of the EML4-ALK transcript, cell viability was decreased up to 50%. Cas13a could effectively knock down FLuc luminescence (70–76%), mCherry fluorescence (72%), and EML4-ALK at the protein (>80%) and transcript levels (26%). Thus, Cas13a has strong potential for use in RNA regulation and therapeutics, and could contribute to the development of personalized medicine.

239 MAC-T CELLS AS A TOOL TO EXAMINE GENOME EDITING USING CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)

2016 ◽  
Vol 28 (2) ◽  
pp. 251
Author(s):  
S. N. Lotti ◽  
I. Tasan ◽  
H. Zhao ◽  
M. B. Wheeler
Keyword(s):  
T Cells ◽  
Milk Production ◽  
Fluorescent Protein ◽  
The United States ◽  
The Other ◽  
Total Efficiency ◽  
Guide Rnas ◽  
Exon 1 ◽  
Green Fluorescent ◽  
A Site

In 2050, the expected size of the human population is 9 billion, the demand for food will increase, and the demand for milk will increase along with it. Genetically modifying animals is a tool that can be used to meet this growing demand. In the United States, Holstein is the leading breed for milk production and Holsteins produce on average 24 291 pounds of milk per year, whereas Jerseys, the other major dairy breed, produce on average 16 997 pounds. Their ability to produce large quantities of milk is linked to 2 mutations. These mutations are on the α-lactalbumin (α-lac) gene; the α-lac exon (+1) corresponds to the transcription start point of α-lac, (+15) and (–1689) are the positions corresponding to the single nucleotide polymorphism associated with increased milk production. Holstein cows have an adenine at both of these positions in contrast to the other cattle breeds with lower milk production, which have either a cytosine or guanine at either position. Inserting an adenine at position (+15) and (–1689) in cows without this mutation could lead to increased milk production and a better response to market demands. The purpose of this experiment was to test the cutting efficiency of candidate clustered regularly interspaced short palindromic repeats (CRISPR) that will later be used in knock-in experiments. CRISPRs were used because the CRISPR-Cas9 system is inexpensive, easily programmed, and efficient. In this preliminary study, we worked with Holstein MAC-T cells, which already contain the mutation at both positions. CRISPRs were used on this cell line to cut the DNA at a site near the mutation. Based on the genomic DNA sequence of these MAC-T cells, 3 guide RNAs were designed. Cells were then transfected with the designed CRISPRs by a variety of transfection methods, including Fugene™ (Promega, Madison, WI, USA), electroporation, and Lipofectamine (ThermoFisher Scientific, Waltham, MA, USA). Green fluorescent protein was used to determine the efficiency of transfection; 30% efficiency was seen for Fugene™, whereas electroporation and Lipofectamine™ had 70% efficiency. To select for successfully transfected cells, puromycin selection was applied. The DNA was later extracted and sent in for sequencing. Next, the website TIDE was used to compare the transfected MAC-T cells to normal MAC-T cells. The TIDE software measures the editing efficiency and looks for major insertions or deletions in pools of DNA by comparing 2 sequences to quantify the editing efficacy of CRISPR-Cas9. Our results showed that CRISPRs successfully cut the DNA near the α-lac mutation region with a total efficiency of 13.8%. The desired next step will be to insert a single-strand oligonucleotide (ssODN) donor to make a single basepair mutation. The ultimate aim of this research would be to insert these mutations into other cattle species in order to increase their milk production.

scholarly journals Design of a polypeptide FRET substrate that facilitates study of the antimicrobial protease lysostaphin

2009 ◽  
Vol 418 (3) ◽  
pp. 615-624 ◽  
Author(s):  
Philip Bardelang ◽  
Mireille Vankemmelbeke ◽  
Ying Zhang ◽  
Hannah Jarvis ◽  
Eleni Antoniadou ◽  
...  
Keyword(s):  
Cell Walls ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Target Site ◽  
Protein Domain ◽  
Serine Residue ◽  
Endopeptidase Activity ◽  
Green Fluorescent ◽  
Colicin E9

We have developed a polypeptide lysostaphin FRET (fluorescence resonance energy transfer) substrate (MV11F) for the endopeptidase activity of lysostaphin. Site-directed mutants of lysostaphin that abolished the killing activity against Staphylococcus aureus also completely inhibited the endopeptidase activity against the MV11 FRET substrate. Lysostaphin-producing staphylococci are resistant to killing by lysostaphin through incorporation of serine residues at positions 3 and 5 of the pentaglycine cross-bridge in their cell walls. The MV11 FRET substrate was engineered to introduce a serine residue at each of four positions of the pentaglycine target site and it was found that only a serine residue at position 3 completely inhibited cleavage. The introduction of random, natural amino acid substitutions at position 3 of the pentaglycine target site demonstrated that only a glycine residue at this position was compatible with lysostaphin cleavage of the MV11 FRET substrate. A second series of polypeptide substrates (decoys) was developed with the GFP (green fluorescent protein) domain of MV11 replaced with that of the DNase domain of colicin E9. Using a competition FRET assay, the lysostaphin endopeptidase was shown to bind to a decoy peptide containing a GGSGG cleavage site. The MV11 substrate provides a valuable system to facilitate structure/function studies of the endopeptidase activity of lysostaphin and its orthologues.

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