scholarly journals Integrative Analysis of CRISPR/Cas9 Target Sites in the HumanHBBGene

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Yumei Luo ◽  
Detu Zhu ◽  
Zhizhuo Zhang ◽  
Yaoyong Chen ◽  
Xiaofang Sun
Keyword(s):  
Rational Design ◽  
Disease Modeling ◽  
Globin Gene ◽  
Transcriptional Factor ◽  
Coding Regions ◽  
Crispr System ◽  
Target Sites ◽  
Artificial Nuclease ◽  
Target Effects ◽  
Target Sets

Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has emerged as a powerful customizable artificial nuclease to facilitate precise genetic correction for tissue regeneration and isogenic disease modeling. However, previous studies reported substantial off-target activities of CRISPR system in human cells, and the enormous putative off-target sites are labor-intensive to be validated experimentally, thus motivating bioinformatics methods for rational design of CRISPR system and prediction of its potential off-target effects. Here, we describe an integrative analytical process to identify specific CRISPR target sites in the humanβ-globin gene (HBB) and predict their off-target effects. Our method includes off-target analysis in both coding and noncoding regions, which was neglected by previous studies. It was found that the CRISPR target sites in the introns have fewer off-target sites in the coding regions than those in the exons. Remarkably, target sites containing certain transcriptional factor motif have enriched binding sites of relevant transcriptional factor in their off-target sets. We also found that the intron sites have fewer SNPs, which leads to less variation of CRISPR efficiency in different individuals during clinical applications. Our studies provide a standard analytical procedure to select specific CRISPR targets for genetic correction.

scholarly journals Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects

2020 ◽  
Vol 6 (29) ◽  
pp. eaba1773 ◽  
Author(s):  
Sangsin Lee ◽  
Ning Ding ◽  
Yidi Sun ◽  
Tanglong Yuan ◽  
Jing Li ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
High Precision ◽  
Disease Modeling ◽  
Human Cells ◽  
Sequencing Analysis ◽  
Injection Method ◽  
Cell Embryo ◽  
Human Apobec3g ◽  
Target Effects

Cytosine base editors (CBEs) enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks. However, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. In the most challenging circumstance, when a bystander C is adjacent to the targeted C, existing base editors fail to discriminate them and edit both Cs. To improve the precision of CBE, we identified and engineered the human APOBEC3G (A3G) deaminase; when fused to the Cas9 nickase, the resulting A3G-BEs exhibit selective editing of the second C in the 5′-CC-3′ motif in human cells. Our A3G-BEs could install a single disease-associated C-to-T substitution with high precision. The percentage of perfectly modified alleles is more than 6000-fold for disease correction and more than 600-fold for disease modeling compared with BE4max. On the basis of the two-cell embryo injection method and RNA sequencing analysis, our A3G-BEs showed minimum genome- and transcriptome-wide off-target effects, achieving high targeting fidelity.

scholarly journals BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
András Tálas ◽  
Dorottya A. Simon ◽  
Péter I. Kulcsár ◽  
Éva Varga ◽  
Sarah L. Krausz ◽  
...  
Keyword(s):  
High Throughput ◽  
Genome Engineering ◽  
Sequence Context ◽  
Base Editing ◽  
Target Dna ◽  
Dna Strands ◽  
Deaminase Domain ◽  
Target Effects ◽  
Target Sets

AbstractAdenine and cytosine base editors (ABE, CBE) allow for precision genome engineering. Here, Base Editor Activity Reporter (BEAR), a plasmid-based fluorescent tool is introduced, which can be applied to report on ABE and CBE editing in a virtually unrestricted sequence context or to label base edited cells for enrichment. Using BEAR-enrichment, we increase the yield of base editing performed by nuclease inactive base editors to the level of the nickase versions while maintaining significantly lower indel background. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we examine whether increased fidelity SpCas9 variants can be used to decrease SpCas9-dependent off-target effects of ABE and CBE. Comparing them on the same target sets reveals that CBE remains active on sequences, where increased fidelity mutations and/or mismatches decrease the activity of ABE. Our results suggest that the deaminase domain of ABE is less effective to act on rather transiently separated target DNA strands, than that of CBE explaining its lower mismatch tolerance.

scholarly journals Microhomologies are prevalent at Cas9-induced larger deletions

2019 ◽  
Vol 47 (14) ◽  
pp. 7402-7417 ◽  
Author(s):  
Dominic D G Owens ◽  
Adam Caulder ◽  
Vincent Frontera ◽  
Joe R Harman ◽  
Alasdair J Allan ◽  
...  
Keyword(s):  
Genome Editing ◽  
Biomedical Research ◽  
High Frequency ◽  
Cultured Cells ◽  
Mouse Embryos ◽  
Deletion Breakpoint ◽  
End Joining ◽  
Crispr System ◽  
Cas9 Nuclease ◽  
Target Sites

Abstract The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.

Two Novel and Five Rare Mutations in the Non Coding Regions of the β-Globin Gene in the Iranian Population

Hemoglobin ◽  
2020 ◽  
Vol 44 (4) ◽  
pp. 225-230
Author(s):  
Mohammad Hamid ◽  
Ebtesam Zargan Nezhad ◽  
Bijan Keikhaei ◽  
Hamid Galehdari ◽  
Alihossein Saberi ◽  
...  
Keyword(s):  
Globin Gene ◽  
Iranian Population ◽  
Coding Regions ◽  
Rare Mutations

A broad activity screen in support of a chemogenomic map for kinase signalling research and drug discovery

2013 ◽  
Vol 451 (2) ◽  
pp. 313-328 ◽  
Author(s):  
Yinghong Gao ◽  
Stephen P. Davies ◽  
Martin Augustin ◽  
Anna Woodward ◽  
Umesh A. Patel ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Drug Discovery ◽  
Small Molecules ◽  
Chemical Structure ◽  
Rational Design ◽  
Kinase Inhibitors ◽  
Biochemical Assays ◽  
Potential Applications ◽  
Specific Inhibitors ◽  
Target Effects

Despite the development of a number of efficacious kinase inhibitors, the strategies for rational design of these compounds have been limited by target promiscuity. In an effort to better understand the nature of kinase inhibition across the kinome, especially as it relates to off-target effects, we screened a well-defined collection of kinase inhibitors using biochemical assays for inhibitory activity against 234 active human kinases and kinase complexes, representing all branches of the kinome tree. For our study we employed 158 small molecules initially identified in the literature as potent and specific inhibitors of kinases important as therapeutic targets and/or signal transduction regulators. Hierarchical clustering of these benchmark kinase inhibitors on the basis of their kinome activity profiles illustrates how they relate to chemical structure similarities and provides new insights into inhibitor specificity and potential applications for probing new targets. Using this broad dataset, we provide a framework for assessing polypharmacology. We not only discover likely off-target inhibitor activities and recommend specific inhibitors for existing targets, but also identify potential new uses for known small molecules.

scholarly journals Role of NF-Y in In Vivo Regulation of the γ-Globin Gene

2001 ◽  
Vol 21 (9) ◽  
pp. 3083-3095 ◽  
Author(s):  
Zhijun Duan ◽  
George Stamatoyannopoulos ◽  
Qiliang Li
Keyword(s):  
Globin Gene ◽  
Gene Promoter ◽  
Transcriptional Factor ◽  
Proximal Promoter ◽  
Transcription Cofactor ◽  
Basal Transcriptional Machinery ◽  
Ccaat Box ◽  
Chromatin Opening

ABSTRACT The duplicated CCAAT box is required for γ gene expression. We report here that the transcriptional factor NF-Y is recruited to the duplicated CCAAT box in vivo. A mutation of the duplicated CCAAT box that severely disrupts the NF-Y binding also reduces the accessibility level of the γ gene promoter, affects the assembly of basal transcriptional machinery, and increases the recruitment of GATA-1 to the locus control region (LCR) and the proximal promoter and the recruitment of transcription cofactor CBP/p300 to the LCR. These findings suggest that recruitment of NF-Y to the duplicated CCAAT box plays a role in the chromatin opening of the γ gene promoter as well as in the communication between the γ gene promoter and the LCR.

The transcriptional factor, IKAROS, plays a key role in γ–β globin gene switching

2007 ◽  
Vol 38 (2) ◽  
pp. 168-169
Author(s):  
J.R. Keys ◽  
P. Papathanasiou ◽  
C.C. Goodnow ◽  
A.C. Perkins
Keyword(s):  
Globin Gene ◽  
Transcriptional Factor ◽  
Gene Switching

scholarly journals Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Luhan Yang ◽  
Dennis Grishin ◽  
Gang Wang ◽  
John Aach ◽  
Cheng-Zhong Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
In Silico ◽  
High Efficiency ◽  
Specific Gene ◽  
Whole Genome ◽  
Single Nucleotide ◽  
Target Sites ◽  
Target Effects

Abstract CRISPR/Cas9 has demonstrated a high-efficiency in site-specific gene targeting. However, potential off-target effects of the Cas9 nuclease represent a major safety concern for any therapeutic application. Here, we knock out the Tafazzin gene by CRISPR/Cas9 in human-induced pluripotent stem cells with 54% efficiency. We combine whole-genome sequencing and deep-targeted sequencing to characterise the off-target effects of Cas9 editing. Whole-genome sequencing of Cas9-modified hiPSC clones detects neither gross genomic alterations nor elevated mutation rates. Deep sequencing of in silico predicted off-target sites in a population of Cas9-treated cells further confirms high specificity of Cas9. However, we identify a single high-efficiency off-target site that is generated by a common germline single-nucleotide variant (SNV) in our experiment. Based on in silico analysis, we estimate a likelihood of SNVs creating off-target sites in a human genome to be ~1.5–8.5%, depending on the genome and site-selection method, but also note that mutations might be generated at these sites only at low rates and may not have functional consequences. Our study demonstrates the feasibility of highly specific clonal ex vivo gene editing using CRISPR/Cas9 and highlights the value of whole-genome sequencing before personalised CRISPR design.

scholarly journals No observable guide-RNA-independent off-target mutation induced by prime editor

2021 ◽  
Author(s):  
Runze Gao ◽  
Zhi-Can Fu ◽  
Xiangyang Li ◽  
Ying Wang ◽  
Jia Wei ◽  
...  
Keyword(s):  
Transcriptome Sequencing ◽  
High Specificity ◽  
Human Cells ◽  
Whole Genome ◽  
Guide Rna ◽  
Genome Wide ◽  
Target Sites ◽  
Whole Transcriptome Sequencing ◽  
Whole Transcriptome ◽  
Target Effects

Prime editor (PE) has been recently developed to induce efficient and precise on-target editing, whereas its guide RNA (gRNA)-independent off-target effects remain unknown. Here, we used whole-genome and whole-transcriptome sequencing to determine gRNA-independent off-target mutations in cells expanded from single colonies, in which PE generated precise editing at on-target sites. We found that PE triggered no observable gRNA-independent off-target mutation genome-wide or transcriptome-wide in transfected human cells, highlighting its high specificity.

scholarly journals In VivoTesting of MicroRNA-Mediated Gene Knockdown in Zebrafish

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Ivone Un San Leong ◽  
Chuan-Ching Lan ◽  
Jonathan R. Skinner ◽  
Andrew N. Shelling ◽  
Donald R. Love
Keyword(s):  
Disease Modeling ◽  
Zebrafish Genome ◽  
Gene Knockdown ◽  
Chemical Mutagenesis ◽  
Vector System ◽  
Orthologous Genes ◽  
Cardiac Disorder ◽  
Qt Syndrome ◽  
Target Effects

The zebrafish (Danio rerio) has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans. The number of tools available to manipulate the zebrafish genome is limited and many currently used techniques are only effective during early development (such as morpholino-based antisense technology) or it is phenotypically driven and does not offer targeted gene knockdown (such as chemical mutagenesis). The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest. In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transientin vivoeGFP sensor assay system. We chose to apply this system to the knockdown of transcripts that are implicated in the human cardiac disorder, Long QT syndrome.

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