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 Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Minh Thuan Nguyen Tran ◽  
Mohd Khairul Nizam Mohd Khalid ◽  
Qi Wang ◽  
Jacqueline K. R. Walker ◽  
Grace E. Lidgerwood ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Editing Efficiency ◽  
Base Editing ◽  
Target Dna ◽  
Dna Editing ◽  
Target Activity ◽  
Adenine Base

Abstract Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.

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

2020 ◽  
Author(s):  
András Tálas ◽  
Dorottya Simon ◽  
Péter Kulcsár ◽  
Éva Varga ◽  
Ervin Welker
Keyword(s):  
High Throughput ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Single Base ◽  
Base Editing ◽  
Target Effects

Abstract Adenine and cytosine base editors (ABE, CBE) are designed to generate single base mutations in genes without necessarily generating DNA double-strand breaks and undesired indel mutations. However, the activity of base editors employing an inactive (dead) SpCas9 is generally low, which may be increased only at the expense of generating undesired indels by using a nickase SpCas9. We have increased the efficiency of dead base editors to a level comparable to that of nickase base editors by enriching cells labelled for efficient base editing using Base Editor Activity Reporter (BEAR), a plasmid-based, fluorescent tool. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we have examined the applicability of increased fidelity variants to decrease Cas9-dependent off-target effects that revealed that CBE remains active on off-targets where increased fidelity mutations and/or mismatches decrease the activity of ABE, making the strategy of applying increased fidelity variants more beneficial for ABE than for CBE.

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 Recent advances in CRISPR/Cas9 and applications for wheat functional genomics and breeding

aBIOTECH ◽  
2021 ◽  
Author(s):  
Jun Li ◽  
Yan Li ◽  
Ligeng Ma
Keyword(s):  
Functional Genomics ◽  
Genome Editing ◽  
Functional Annotation ◽  
Genome Engineering ◽  
Agronomic Traits ◽  
Wheat Breeding ◽  
Breeding Programs ◽  
Base Editing ◽  
The World ◽  
World Food Security

AbstractCommon wheat (Triticum aestivum L.) is one of the three major food crops in the world; thus, wheat breeding programs are important for world food security. Characterizing the genes that control important agronomic traits and finding new ways to alter them are necessary to improve wheat breeding. Functional genomics and breeding in polyploid wheat has been greatly accelerated by the advent of several powerful tools, especially CRISPR/Cas9 genome editing technology, which allows multiplex genome engineering. Here, we describe the development of CRISPR/Cas9, which has revolutionized the field of genome editing. In addition, we emphasize technological breakthroughs (e.g., base editing and prime editing) based on CRISPR/Cas9. We also summarize recent applications and advances in the functional annotation and breeding of wheat, and we introduce the production of CRISPR-edited DNA-free wheat. Combined with other achievements, CRISPR and CRISPR-based genome editing will speed progress in wheat biology and promote sustainable agriculture.

scholarly journals Optimization of C-to-G base editors with sequence context preference predictable by machine learning methods

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tanglong Yuan ◽  
Nana Yan ◽  
Tianyi Fei ◽  
Jitan Zheng ◽  
Juan Meng ◽  
...  
Keyword(s):  
Machine Learning ◽  
High Efficiency ◽  
Codon Optimization ◽  
Sequence Context ◽  
Learning Methods ◽  
Specific Sequence ◽  
Uracil Dna Glycosylase ◽  
Base Editing ◽  
Machine Learning Methods ◽  
Deep Learning Model

AbstractEfficient and precise base editors (BEs) for C-to-G transversion are highly desirable. However, the sequence context affecting editing outcome largely remains unclear. Here we report engineered C-to-G BEs of high efficiency and fidelity, with the sequence context predictable via machine-learning methods. By changing the species origin and relative position of uracil-DNA glycosylase and deaminase, together with codon optimization, we obtain optimized C-to-G BEs (OPTI-CGBEs) for efficient C-to-G transversion. The motif preference of OPTI-CGBEs for editing 100 endogenous sites is determined in HEK293T cells. Using a sgRNA library comprising 41,388 sequences, we develop a deep-learning model that accurately predicts the OPTI-CGBE editing outcome for targeted sites with specific sequence context. These OPTI-CGBEs are further shown to be capable of efficient base editing in mouse embryos for generating Tyr-edited offspring. Thus, these engineered CGBEs are useful for efficient and precise base editing, with outcome predictable based on sequence context of targeted sites.

scholarly journals Learning the heterogeneous hypermutation landscape of immunoglobulins from high-throughput repertoire data

2020 ◽  
Author(s):  
Natanael Spisak ◽  
Aleksandra M. Walczak ◽  
Thierry Mora
Keyword(s):  
B Cell ◽  
High Throughput ◽  
Affinity Maturation ◽  
B Cell Receptors ◽  
Absolute Position ◽  
Sequence Context ◽  
Cell Lineages ◽  
Selective Forces ◽  
Antigenic Targets ◽  
Complementarity Determining Region

Somatic hypermutations of immunoglobulin (Ig) genes occurring during affinity maturation drive B-cell receptors’ ability to evolve strong binding to their antigenic targets. The landscape of these mutations is highly heterogeneous, with certain regions of the Ig gene being preferentially targeted. However, a rigorous quantification of this bias has been difficult because of phylogenetic correlations between sequences and the interference of selective forces. Here, we present an approach that corrects for these issues, and use it to learn a model of hypermutation preferences from a recently published large IgH repertoire dataset. The obtained model predicts mutation profiles accurately and in a reproducible way, including in the previously uncharacterized Complementarity Determining Region 3, revealing that both the sequence context of the mutation and its absolute position along the gene are important. In addition, we show that hypermutations occurring concomittantly along B-cell lineages tend to co-localize, suggesting a possible mechanism for accelerating affinity maturation.

scholarly journals Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors

2018 ◽  
Author(s):  
Shama Sograte-Idrissi ◽  
Nazar Oleksiievets ◽  
Sebastian Isbaner ◽  
Mariana Eggert-Martinez ◽  
Jörg Enderlein ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Super Resolution ◽  
Resolving Power ◽  
Acquisition Time ◽  
Efficient Manner ◽  
Target Dna ◽  
Multiplexed Imaging ◽  
Dna Strands ◽  
Linkage Error ◽  
20 Nm

AbstractDNA-PAINT is a rapidly developing fluorescence super-resolution technique which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20-25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using 3 nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the 3 targets with 20 nm resolution, and within only 35 minutes acquisition time.

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