scholarly journals Transient overexpression of DNA adenine methylase enables efficient and mobile genome engineering with reduced off-target effects

2015 ◽  
Vol 44 (4) ◽  
pp. e36-e36 ◽  
Author(s):  
Rebecca M. Lennen ◽  
Annika I. Nilsson Wallin ◽  
Margit Pedersen ◽  
Mads Bonde ◽  
Hao Luo ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Dna Adenine Methylase ◽  
Transient Overexpression ◽  
Target Effects

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.

Recent advances in DNA-free editing and precise base editing in plants

2017 ◽  
Vol 1 (2) ◽  
pp. 161-168 ◽  
Author(s):  
Yi Zhang ◽  
Caixia Gao
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Point Mutations ◽  
Plant Genome ◽  
The Novel ◽  
Future Perspectives ◽  
Delivery Methods ◽  
Base Editing ◽  
Short Palindromic Repeat ◽  
Target Effects

Genome-editing technologies based on the CRISPR (clustered regularly interspaced short palindromic repeat) system have been widely used in plants to investigate gene function and improve crop traits. The recently developed DNA-free delivery methods and precise base-editing systems provide new opportunities for plant genome engineering. In this review, we describe the novel DNA-free genome-editing methods in plants. These methods reduce off-target effects and may alleviate regulatory concern about genetically modified plants. We also review applications of base-editing systems, which are highly effective in generating point mutations and are of great value for introducing agronomically valuable traits. Future perspectives for DNA-free editing and base editing are also discussed.

scholarly journals Genome editing in mammals using CRISPR type I-D nuclease

2020 ◽  
Author(s):  
Keishi Osakabe ◽  
Naoki Wada ◽  
Emi Murakami ◽  
Yuriko Osakabe
Keyword(s):  
Genome Editing ◽  
Genomic Dna ◽  
Genome Engineering ◽  
Dna Degradation ◽  
Targeted Mutagenesis ◽  
Eukaryotic Cells ◽  
Type I ◽  
Effector Pathway ◽  
Target Dna ◽  
Target Effects

SUMMARYAdoption of the CRISPR-Cas system has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I—the most abundant CRISPR system in bacteria—has been less developed. Type I systems in which Cas3 nuclease degrades the target DNA are known; in contrast, for the sub-type CRISPR type I-D (TiD), which lacks a typical Cas3 nuclease in its cascade, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d—a nuclease in TiD—is multi-functional in PAM recognition, stabilization and target DNA degradation. TiD can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. TiD off-target effects, which were dependent on the mismatch position in the protospacer of TiD, were also identified. Our findings suggest TiD as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.

scholarly journals Precision Genome and Base Editing by Regulating mRNA Nuclear Export Using Selective Inhibitors of Nuclear Export (SINEs)

2021 ◽  
Author(s):  
Yan-ru Cui ◽  
Shao-jie Wang ◽  
Tiancheng Ma ◽  
Peihong Yu ◽  
Jun Chen ◽  
...  
Keyword(s):  
Anticancer Drug ◽  
Small Molecule ◽  
Nuclear Export ◽  
Genome Engineering ◽  
Cellular Activity ◽  
Selective Inhibitors ◽  
Base Editing ◽  
Cas9 Protein ◽  
Target Effects ◽  
Cas9 Mrna

AbstractCRISPR-based genome engineering tools are associated with off-target effects that constitutively active Cas9 protein may instigate. In the present study, we screened for irreversible small molecule off-switches of CRISPR-Cas9 and discovered that selective inhibitors of nuclear export (SINEs) could inhibit the cellular activity of CRISPR-Cas9 by interfering with the nuclear export of Cas9 mRNA. We subsequently found that SINEs, including an FDA-approved anticancer drug KPT330, could improve the specificities of CRISPR-Cas9-based genome and base editing tools in human cells.

scholarly journals Unified energetics analysis unravels SpCas9 cleavage activity for optimal gRNA design

2019 ◽  
Vol 116 (18) ◽  
pp. 8693-8698 ◽  
Author(s):  
Dong Zhang ◽  
Travis Hurst ◽  
Dongsheng Duan ◽  
Shi-Jie Chen
Keyword(s):  
Genome Engineering ◽  
Learning Approaches ◽  
Loop Formation ◽  
Unified Framework ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cleavage Activity ◽  
Protospacer Adjacent Motif ◽  
Traditional Approaches ◽  
Target Effects

While CRISPR/Cas9 is a powerful tool in genome engineering, the on-target activity and off-target effects of the system widely vary because of the differences in guide RNA (gRNA) sequences and genomic environments. Traditional approaches rely on separate models and parameters to treat on- and off-target cleavage activities. Here, we demonstrate that a free-energy scheme dominates the Cas9 editing efficacy and delineate a method that simultaneously considers on-target activities and off-target effects. While data-driven machine-learning approaches learn rules to model particular datasets, they may not be as transferrable to new systems or capable of producing new mechanistic insights as principled physical approaches. By integrating the energetics of R-loop formation under Cas9 binding, the effect of the protospacer adjacent motif sequence, and the folding stability of the whole single guide RNA, we devised a unified, physical model that can apply to any cleavage-activity dataset. This unified framework improves predictions for both on-target activities and off-target efficiencies of spCas9 and may be readily transferred to other systems with different guide RNAs or Cas9 ortholog proteins.

Perfecting Targeting in CRISPR

2021 ◽  
Vol 55 (1) ◽  
Author(s):  
Hainan Zhang ◽  
Tong Li ◽  
Yidi Sun ◽  
Hui Yang
Keyword(s):  
Rna Editing ◽  
Online Publication ◽  
Genome Engineering ◽  
Basic Research ◽  
Annual Review ◽  
Publication Date ◽  
Full Potential ◽  
Dna And Rna ◽  
Genome Wide ◽  
Target Effects

CRISPR-based genome editing holds promise for genome engineering and other applications in diverse organisms. Defining and improving the genome-wide and transcriptome-wide specificities of these editing tools are essential for realizing their full potential in basic research and biomedical therapeutics. This review provides an overview of CRISPR-based DNA- and RNA-editing technologies, methods to quantify their specificities, and key solutions to reduce off-target effects for research and improve therapeutic applications. Expected final online publication date for the Annual Review of Genetics, Volume 55 is November 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Krzysztof Chylinski ◽  
Maria Hubmann ◽  
Ruth E. Hanna ◽  
Connor Yanchus ◽  
Georg Michlits ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Gene Editing ◽  
Genome Engineering ◽  
Expression Cassette ◽  
Rapid Induction ◽  
Versatile Tool ◽  
Cre Recombination ◽  
Rapid Switching ◽  
Target Effects

AbstractCRISPR-Cas9 is an efficient and versatile tool for genome engineering in many species. However, inducible CRISPR-Cas9 editing systems that regulate Cas9 activity or sgRNA expression often suffer from significant limitations, including reduced editing capacity, off-target effects, or leaky expression. Here, we develop a precisely controlled sgRNA expression cassette that can be combined with widely-used Cre systems, termed CRISPR-Switch (SgRNA With Induction/Termination by Cre Homologous recombination). Switch-ON facilitates controlled, rapid induction of sgRNA activity. In turn, Switch-OFF-mediated termination of editing improves generation of heterozygous genotypes and can limit off-target effects. Furthermore, we design sequential CRISPR-Switch-based editing of two loci in a strictly programmable manner and determined the order of mutagenic events that leads to development of glioblastoma in mice. Thus, CRISPR-Switch substantially increases the versatility of gene editing through precise and rapid switching ON or OFF sgRNA activity, as well as switching OVER to secondary sgRNAs.

scholarly journals Off-target Effects in CRISPR/Cas9-mediated Genome Engineering

2015 ◽  
Vol 4 ◽  
pp. e264 ◽  
Author(s):  
Xiao-Hui Zhang ◽  
Louis Y Tee ◽  
Xiao-Gang Wang ◽  
Qun-Shan Huang ◽  
Shi-Hua Yang
Keyword(s):  
Genome Engineering ◽  
Target Effects

CRISPR-Cas9 genome engineering: trends in medicine and health

2021 ◽  
Vol 21 ◽  
Author(s):  
Sumera Zaib ◽  
Mushtaq Saleem ◽  
Imtiaz Khan
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
Genetic Manipulation ◽  
Engineering Process ◽  
High Cholesterol ◽  
The Past ◽  
Current Review ◽  
Medical Researchers ◽  
Short Palindromic Repeat ◽  
Target Effects

: The ability to engineer biological systems and organisms holds enormous potential for applications across basic science, medicine and biotechnology. Over the past few decades, the development of CRISPR (clustered regularly interspaced short palindromic repeat) has revolutionized the whole genetic engineering process utilizing the principles of Watson-Crick base pairing. CRISPR-Cas9 technology offers the simplest, fastest, most versatile, reliable and precise method of genetic manipulation thus enabling geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence. The current review focuses on the applications of CRISPR-Cas9 in the field of medical research. Compared with other gene editing technologies, CRISPR/Cas9 demonstrates numerous advantages for the treatment of various medical conditions including cancer, hepatitis B, cardiovascular diseases or even high cholesterol. Given its promising performance, CRISPR/Cas9 gene editing technology will surely help in the therapy of several disorders while addressing the issues pertaining to the minimization of the off-target effects of gene editing and incomplete matches between sgRNA and genomic DNA by Cas9.

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