scholarly journals Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments

2019 ◽  
Author(s):  
Hera Canaj ◽  
Jeffrey A. Hussmann ◽  
Han Li ◽  
Kyle A. Beckman ◽  
Leeanne Goodrich ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Cell Types ◽  
Strand Break ◽  
Homology Directed Repair ◽  
Wide Range ◽  
Monitoring Accuracy ◽  
Long Read ◽  
Repair Mechanisms ◽  
Comprehensive Framework ◽  
Two Sides

AbstractCRISPR/Cas technologies have transformed our ability to add functionality to the genome by knock-in of payload via homology-directed repair (HDR). However, a systematic and quantitative profiling of the knock-in integration landscape is still lacking. Here, we present a framework based on long-read sequencing and an integrated computational pipeline (knock-knock) to analyze knock-in repair outcomes across a wide range of experimental parameters. Our data uncover complex repair profiles, with perfect HDR often accounting for a minority of payload integration events, and reveal markedly distinct mis-integration patterns between cell-types or forms of HDR templates used. Our analysis demonstrates that the two sides of a given double-strand break can be repaired by separate pathways and identifies a major role for sequence micro-homology in driving donor mis-integration. Altogether, our comprehensive framework paves the way for investigating repair mechanisms, monitoring accuracy, and optimizing the precision of genome engineering.

Double strand break (DSB) repair pathways in plants and their application in genome engineering

2021 ◽  
pp. 27-62
Author(s):  
Natalja Beying ◽  
◽  
Carla Schmidt ◽  
Holger Puchta ◽  
◽  
...  
Keyword(s):  
Genome Engineering ◽  
Plant Cells ◽  
Strand Break ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Repair Mechanisms ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
Underlying Mechanisms

In genome engineering, after targeted induction of double strand breaks (DSBs) researchers take advantage of the organisms’ own repair mechanisms to induce different kinds of sequence changes into the genome. Therefore, understanding of the underlying mechanisms is essential. This chapter will review in detail the two main pathways of DSB repair in plant cells, non-homologous end joining (NHEJ) and homologous recombination (HR) and sum up what we have learned over the last decades about them. We summarize the different models that have been proposed and set these into relation with the molecular outcomes of different classes of DSB repair. Moreover, we describe the factors that have been identified to be involved in these pathways. Applying this knowledge of DSB repair should help us to improve the efficiency of different types of genome engineering in plants.

scholarly journals Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Beau R. Webber ◽  
Cara-lin Lonetree ◽  
Mitchell G. Kluesner ◽  
Matthew J. Johnson ◽  
Emily J. Pomeroy ◽  
...  
Keyword(s):  
T Cells ◽  
Genome Engineering ◽  
Cellular Therapy ◽  
Cell Types ◽  
Strand Break ◽  
Double Strand Break ◽  
Adoptive Cellular Therapy ◽  
Highly Efficient ◽  
Human T Cell ◽  
T Cell Engineering

AbstractThe fusion of genome engineering and adoptive cellular therapy holds immense promise for the treatment of genetic disease and cancer. Multiplex genome engineering using targeted nucleases can be used to increase the efficacy and broaden the application of such therapies but carries safety risks associated with unintended genomic alterations and genotoxicity. Here, we apply base editor technology for multiplex gene modification in primary human T cells in support of an allogeneic CAR-T platform and demonstrate that base editor can mediate highly efficient multiplex gene disruption with minimal double-strand break induction. Importantly, multiplex base edited T cells exhibit improved expansion and lack double strand break-induced translocations observed in T cells edited with Cas9 nuclease. Our findings highlight base editor as a powerful platform for genetic modification of therapeutically relevant primary cell types.

scholarly journals Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair

2014 ◽  
Vol 111 (10) ◽  
pp. E924-E932 ◽  
Author(s):  
Luther Davis ◽  
Nancy Maizels
Keyword(s):  
Genome Engineering ◽  
Alternative Pathway ◽  
Strand Break ◽  
End Joining ◽  
Single Strand Break ◽  
Homology Directed Repair ◽  
Single Strand Break Repair ◽  
Efficient Alternative ◽  
Break Repair ◽  
Dna Nicks

DNA nicks are the most common form of DNA damage, and if unrepaired can give rise to genomic instability. In human cells, nicks are efficiently repaired via the single-strand break repair pathway, but relatively little is known about the fate of nicks not processed by that pathway. Here we show that homology-directed repair (HDR) at nicks occurs via a mechanism distinct from HDR at double-strand breaks (DSBs). HDR at nicks, but not DSBs, is associated with transcription and is eightfold more efficient at a nick on the transcribed strand than at a nick on the nontranscribed strand. HDR at nicks can proceed by a pathway dependent upon canonical HDR factors RAD51 and BRCA2; or by an efficient alternative pathway that uses either ssDNA or nicked dsDNA donors and that is strongly inhibited by RAD51 and BRCA2. Nicks generated by either I-AniI or the CRISPR/Cas9D10A nickase are repaired by the alternative HDR pathway with little accompanying mutagenic end-joining, so this pathway may be usefully applied to genome engineering. These results suggest that alternative HDR at nicks may be stimulated in physiological contexts in which canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated, which occurs frequently in tumors.

scholarly journals Mapping the Genetic Landscape of DNA Double-strand Break Repair

2021 ◽  
Author(s):  
Jeffrey A Hussmann ◽  
Jia Ling ◽  
Purnima Ravisankar ◽  
Jun Yan ◽  
Ann Cirincione ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Strand Break ◽  
Dna Lesions ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Homology Directed Repair ◽  
Genetic Perturbations ◽  
Genetic Landscape ◽  
Repair Mechanisms

Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways that are critical for maintaining genomic integrity. Here we present Repair-seq, a high-throughput screening approach that measures the effects of thousands of genetic perturbations on the distribution of mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair outcomes induced by two programmable nucleases (Cas9 and Cas12a) after knockdown of 476 genes involved in DSB repair or associated processes in the presence or absence of oligonucleotides for homology-directed repair (HDR). The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. Systematic interrogation of these dependencies then uncovered unexpected relationships among DSB repair genes and isolated incompletely characterized repair mechanisms. This work provides a foundation for understanding the complex pathways of DSB repair and for optimizing genome editing across modalities.

scholarly journals New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
Author(s):  
Valmik K. Vyas ◽  
G. Guy Bushkin ◽  
Douglas A. Bernstein ◽  
Matthew A. Getz ◽  
Magdalena Sewastianik ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Genome Engineering ◽  
High Efficiency ◽  
Selection Marker ◽  
Important Species ◽  
Content Type ◽  
Guide Rna ◽  
Wide Range ◽  
Repair Mechanisms ◽  
The Creation

ABSTRACT We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans , Saccharomyces cerevisiae , Candida glabrata , and Naumovozyma castellii . These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms. IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata , as well as in the related yeasts S. cerevisiae and N. castellii . The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.

scholarly journals A genetically encoded inhibitor of 53BP1 to stimulate homology-based gene editing

2016 ◽  
Author(s):  
Marella D. Canny ◽  
Leo C.K. Wan ◽  
Amélie Fradet-Turcotte ◽  
Alexandre Orthwein ◽  
Nathalie Moatti ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Cell Types ◽  
Strand Break ◽  
End Joining ◽  
Dsb Repair ◽  
Non Homologous End Joining ◽  
G1 Cells ◽  
Programmable Nucleases ◽  
End Resection

AbstractThe expanding repertoire of programmable nucleases such as Cas9 brings new opportunities in genetic medicine1–3. In many cases, these nucleases are engineered to induce a DNA double-strand break (DSB) to stimulate precise genome editing by homologous recombination (HR). However, HR efficiency is nearly always hindered by competing DSB repair pathways such as non-homologous end-joining (NHEJ). HR is also profoundly suppressed in non-replicating cells, thus precluding the use of homology-based genome engineering in a wide variety4 of cell types. Here, we report the development of a genetically encoded inhibitor of 53BP1 (known as TP53BP1), a regulator of DSB repair pathway choice5. 53BP1 promotes NHEJ over HR by suppressing end resection, the formation of 3-prime single-stranded DNA tails, which is the rate-limiting step in HR initiation. 53BP1 also blocks the recruitment of the HR factor BRCA1 to DSB sites in G1 cells4,6. The inhibitor of 53BP1 (or i53) was identified through the screening of a massive combinatorial library of engineered ubiquitin variants by phage display7. i53 binds and occludes the ligand binding site of the 53BP1 Tudor domain with high affinity and selectivity, blocking its ability to accumulate at sites of DNA damage. i53 is a potent selective inhibitor of 53BP1 and enhances gene targeting and chromosomal gene conversion, two HR-dependent reactions. Finally, i53 can also activate HR in G1 cells when combined with the activation of end-resection and KEAP1 inhibition. We conclude that 53BP1 inhibition is a robust tool to enhance precise genome editing by canonical HR pathways.

scholarly journals Efficient Immune Cell Genome Engineering with Improved CRISPR Editing Tools

2020 ◽  
Author(s):  
Waipan Chan ◽  
Rachel A. Gottschalk ◽  
Yikun Yao ◽  
Joel L. Pomerantz ◽  
Ronald N. Germain
Keyword(s):  
Immune System ◽  
Genome Editing ◽  
Genome Engineering ◽  
Immune Cell ◽  
Animal Experiments ◽  
Adaptive Immune System ◽  
Cell Types ◽  
Acid Activation ◽  
Homology Directed Repair ◽  
Cell Genome

AbstractCRISPR (clustered regularly interspaced short palindromic repeats)-based methods have revolutionized genome engineering and the study of gene-phenotype relationships. However, modifying cells of the innate immune system, especially macrophages, has been challenging because of cell pathology and low targeting efficiency resulting from nucleic acid activation of sensitive intracellular sensors. Likewise, lymphocytes of the adaptive immune system are largely refractory to CRISPR-enhanced homology-directed repair (HDR) due to inefficient or toxic delivery of donor templates via transient transfection methods. To overcome these challenges and limitations, we developed three improved methods for CRISPR-based genome editing using a hit-and-run transient expression strategy to minimize off-target effects and generate more precise genome editing. Overall, our enhanced CRISPR tools and strategies designed to tackle both murine and human immune cell genome engineering are expected to be widely applicable not only in hematopoietic cells but also other mammalian cell types of interest.All animal experiments were done in accordance with the guidelines of the NIAID/NIH Institutional Animal Care and Use Committee.

scholarly journals Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Natasa Savic ◽  
Femke CAS Ringnalda ◽  
Helen Lindsay ◽  
Christian Berk ◽  
Katja Bargsten ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cell Types ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Covalent Linkage ◽  
Homology Directed Repair ◽  
Cas9 Nuclease ◽  
Repair Mechanisms ◽  
Multiple Cell ◽  
Donor Template

The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, however, leads to relatively low rates of precise editing from donor DNA. Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold, and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus. Enhanced correction rates were observed in multiple cell types and on different genomic loci, suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired.

scholarly journals CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing

2017 ◽  
Author(s):  
Miguel A. Moreno-Mateos ◽  
Juan P. Fernandez ◽  
Romain Rouet ◽  
Maura A. Lane ◽  
Charles E. Vejnar ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genomic Dna ◽  
Genome Engineering ◽  
Lower Efficiency ◽  
Dna Integration ◽  
Homology Directed Repair ◽  
Ribonucleoprotein Complexes ◽  
Wide Range ◽  
Temperature Controlled

Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows post-translational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species.

scholarly journals Efficient photoactivatable Dre recombinase for cell type-specific spatiotemporal control of genome engineering in the mouse

2020 ◽  
Vol 117 (52) ◽  
pp. 33426-33435
Author(s):  
Huiying Li ◽  
Qiansen Zhang ◽  
Yiran Gu ◽  
Yingyin Wu ◽  
Yamei Wang ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Cell Types ◽  
Specific Cell ◽  
Light Illumination ◽  
Cell Type ◽  
Reading Frame ◽  
Chemical Inducers ◽  
Mouse Tissues ◽  
Wide Range ◽  
Cell Type Specific

Precise genetic engineering in specific cell types within an intact organism is intriguing yet challenging, especially in a spatiotemporal manner without the interference caused by chemical inducers. Here we engineered a photoactivatable Dre recombinase based on the identification of an optimal split site and demonstrated that it efficiently regulated transgene expression in mouse tissues spatiotemporally upon blue light illumination. Moreover, through a double-floxed inverted open reading frame strategy, we developed a Cre-activated light-inducible Dre (CALID) system. Taking advantage of well-defined cell-type–specific promoters or a well-established Cre transgenic mouse strain, we demonstrated that the CALID system was able to activate endogenous reporter expression for either bulk or sparse labeling of CaMKIIα-positive excitatory neurons and parvalbumin interneurons in the brain. This flexible and tunable system could be a powerful tool for the dissection and modulation of developmental and genetic complexity in a wide range of biological systems.

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