scholarly journals Engineering a far-red light–activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors

2020 ◽  
Vol 6 (28) ◽  
pp. eabb1777 ◽  
Author(s):  
Yuanhuan Yu ◽  
Xin Wu ◽  
Ningzi Guan ◽  
Jiawei Shao ◽  
Huiying Li ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Light Emitting Diode ◽  
Red Light ◽  
Tumor Model ◽  
End Joining ◽  
Internal Organs ◽  
Fast System ◽  
Homology Directed Repair ◽  
Multiple Loci

It is widely understood that CRISPR-Cas9 technology is revolutionary, with well-recognized issues including the potential for off-target edits and the attendant need for spatiotemporal control of editing. Here, we describe a far-red light (FRL)–activated split-Cas9 (FAST) system that can robustly induce gene editing in both mammalian cells and mice. Through light-emitting diode–based FRL illumination, the FAST system can efficiently edit genes, including nonhomologous end joining and homology-directed repair, for multiple loci in human cells. Further, we show that FAST readily achieves FRL-induced editing of internal organs in tdTomato reporter mice. Finally, FAST was demonstrated to achieve FRL-triggered editing of the PLK1 oncogene in a mouse xenograft tumor model. Beyond extending the spectrum of light energies in optogenetic toolbox for CRISPR-Cas9 technologies, this study demonstrates how FAST system can be deployed for programmable deep tissue gene editing in both biological and biomedical contexts toward high precision and spatial specificity.

scholarly journals Homology Directed Repair by Cas9:Donor Co-localization in Mammalian Cells

2018 ◽  
Author(s):  
Philip J.R. Roche ◽  
Heidi Gytz ◽  
Faiz Hussain ◽  
Christopher J.F. Cameron ◽  
Denis Paquette ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Low Frequency ◽  
Cost Effective ◽  
Hek293 Cells ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

AbstractHomology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPR/Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered Cas9 protein fused to a high-affinity monoavidin domain to deliver biotinylated donor DNA to a DSB site. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9 RNP-donor DNA complex into the cell. Combining these strategies improved HDR percentages of up to 90% in three tested loci (CXCR4, EMX1, and TLR) in standard HEK293 cells. Our approach offers a cost effective, simple and broadly applicable gene editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox.SummaryPrecision gene editing occurs at a low percentage in mammalian cells using Cas9. Colocalization of donor with Cas9MAV and PEI delivery raises HDR occurrence.

scholarly journals Methods Favoring Homology-Directed Repair Choice in Response to CRISPR/Cas9 Induced-Double Strand Breaks

2020 ◽  
Vol 21 (18) ◽  
pp. 6461
Author(s):  
Han Yang ◽  
Shuling Ren ◽  
Siyuan Yu ◽  
Haifeng Pan ◽  
Tingdong Li ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Initial Step ◽  
Double Strand Breaks ◽  
Repair Pathway ◽  
End Joining ◽  
Strand Breaks ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Repair Pathways

Precise gene editing is—or will soon be—in clinical use for several diseases, and more applications are under development. The programmable nuclease Cas9, directed by a single-guide RNA (sgRNA), can introduce double-strand breaks (DSBs) in target sites of genomic DNA, which constitutes the initial step of gene editing using this novel technology. In mammals, two pathways dominate the repair of the DSBs—nonhomologous end joining (NHEJ) and homology-directed repair (HDR)—and the outcome of gene editing mainly depends on the choice between these two repair pathways. Although HDR is attractive for its high fidelity, the choice of repair pathway is biased in a biological context. Mammalian cells preferentially employ NHEJ over HDR through several mechanisms: NHEJ is active throughout the cell cycle, whereas HDR is restricted to S/G2 phases; NHEJ is faster than HDR; and NHEJ suppresses the HDR process. This suggests that definitive control of outcome of the programmed DNA lesioning could be achieved through manipulating the choice of cellular repair pathway. In this review, we summarize the DSB repair pathways, the mechanisms involved in choice selection based on DNA resection, and make progress in the research investigating strategies that favor Cas9-mediated HDR based on the manipulation of repair pathway choice to increase the frequency of HDR in mammalian cells. The remaining problems in improving HDR efficiency are also discussed. This review should facilitate the development of CRISPR/Cas9 technology to achieve more precise gene editing.

scholarly journals Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in Leishmania

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Wen-Wei Zhang ◽  
Greg Matlashewski
Keyword(s):  
Gene Targeting ◽  
Mammalian Cells ◽  
Gene Editing ◽  
Single Strand ◽  
End Joining ◽  
Content Type ◽  
Single Strand Annealing ◽  
Dna Break ◽  
Strand Annealing ◽  
Double Strand Dna

ABSTRACT CRISPR-Cas9 genome editing relies on an efficient double-strand DNA break (DSB) and repair. Contrary to mammalian cells, the protozoan parasite Leishmania lacks the most efficient nonhomologous end-joining pathway and uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair to repair DSBs. Here, we reveal that Leishmania predominantly uses single-strand annealing (SSA) (>90%) instead of MMEJ (<10%) for DSB repair (DSBR) following CRISPR targeting of the miltefosine transporter gene, resulting in 9-, 18-, 20-, and 29-kb sequence deletions and multiple gene codeletions. Strikingly, when targeting the Leishmania donovani LdBPK_241510 gene, SSA even occurred by using direct repeats 77 kb apart, resulting in the codeletion of 15 Leishmania genes, though with a reduced frequency. These data strongly indicate that DSBR is not efficient in Leishmania, which explains why more than half of DSBs led to cell death and why the CRISPR gene-targeting efficiency is low compared with that in other organisms. Since direct repeat sequences are widely distributed in the Leishmania genome, we predict that many DSBs created by CRISPR are repaired by SSA. It is also revealed that DNA polymerase theta is involved in both MMEJ and SSA in Leishmania. Collectively, this study establishes that DSBR mechanisms and their competence in an organism play an important role in determining the outcome and efficacy of CRISPR gene targeting. These observations emphasize the use of donor DNA templates to improve gene editing specificity and efficiency in Leishmania. In addition, we developed a novel Staphylococcus aureus Cas9 constitutive expression vector (pLdSaCN) for gene targeting in Leishmania. IMPORTANCE Due to differences in double-strand DNA break (DSB) repair mechanisms, CRISPR-Cas9 gene editing efficiency can vary greatly in different organisms. In contrast to mammalian cells, the protozoan parasite Leishmania uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair (HDR) to repair DSBs but lacks the nonhomologous end-joining pathway. Here, we show that Leishmania predominantly uses single-strand annealing (SSA) instead of MMEJ for DSB repairs (DSBR), resulting in large deletions that can include multiple genes. This strongly indicates that the overall DSBR in Leishmania is inefficient and therefore can influence the outcome of CRISPR-Cas9 gene editing, highlighting the importance of using a donor DNA to improve gene editing fidelity and efficiency in Leishmania.

scholarly journals Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells

2015 ◽  
Vol 33 (5) ◽  
pp. 543-548 ◽  
Author(s):  
Van Trung Chu ◽  
Timm Weber ◽  
Benedikt Wefers ◽  
Wolfgang Wurst ◽  
Sandrine Sander ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Homology Directed Repair

scholarly journals Targeted Gene Editing in Porcine Spermatogonia

2021 ◽  
Vol 11 ◽  
Author(s):  
Dennis Webster ◽  
Alla Bondareva ◽  
Staci Solin ◽  
Taylor Goldsmith ◽  
Lin Su ◽  
...  
Keyword(s):  
Stem Cell ◽  
Regenerative Medicine ◽  
Human Disease ◽  
Gene Editing ◽  
Disease Modeling ◽  
Germline Stem Cell ◽  
End Joining ◽  
Homology Directed Repair ◽  
Pig Genome ◽  
Targeted Gene Editing

To study the pathophysiology of human diseases, develop innovative treatments, and refine approaches for regenerative medicine require appropriate preclinical models. Pigs share physiologic and anatomic characteristics with humans and are genetically more similar to humans than are mice. Genetically modified pigs are essential where rodent models do not mimic the human disease phenotype. The male germline stem cell or spermatogonial stem cell (SSC) is unique; it is the only cell type in an adult male that divides and contributes genes to future generations, making it an ideal target for genetic modification. Here we report that CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing in porcine spermatogonia that include SSCs is significantly more efficient than previously reported editing with TALENs and allows precise gene editing by homology directed repair (HDR). We also established homology-mediated end joining (HMEJ) as a second approach to targeted gene editing to enable introduction of larger transgenes and/or humanizing parts of the pig genome for disease modeling or regenerative medicine. In summary, the approaches established in the current study result in efficient targeted genome editing in porcine germ cells for precise replication of human disease alleles.

scholarly journals Understanding the diversity of genetic outcomes from CRISPR-Cas generated homology-directed repair

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Brett M. Sansbury ◽  
Amanda M. Hewes ◽  
Eric B. Kmiec
Keyword(s):  
Dna Repair ◽  
Enzymatic Activity ◽  
Broad Spectrum ◽  
Gene Editing ◽  
Cell Free Extract ◽  
End Joining ◽  
Homology Directed Repair ◽  
Editing Activity ◽  
Non Homologous End Joining

AbstractAs CRISPR-Cas systems advance toward clinical application, it is essential to identify all the outcomes of gene-editing activity in human cells. Reports highlighting the remarkable success of homology-directed repair (HDR) in the treatment of inherited diseases may inadvertently underreport the collateral activity of this remarkable technology. We are utilizing an in vitro gene-editing system in which a CRISPR-Cas complex provides the double-stranded cleavage and a mammalian cell-free extract provides the enzymatic activity to promote non-homologous end joining, micro-homology mediated end joining, and homology-directed repair. Here, we detail the broad spectrum of gene-editing reaction outcomes utilizing Cas9 and Cas12a in combination with single-stranded donor templates of the sense and nonsense polarity. This system offers the opportunity to see the range of outcomes of gene-editing reactions in an unbiased fashion, detailing the distribution of DNA repair outcomes as a function of a set of genetic tools.

scholarly journals CRISPR-Cas9: A Genome Editing Tool in Crop Plants: A Review

2021 ◽  
Author(s):  
Varsha Kumari ◽  
Priyanka Kumawat ◽  
Sharanabasappa Yeri ◽  
Shyam Singh Rajput
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Crop Improvement ◽  
End Joining ◽  
Homology Directed Repair ◽  
Ligase Iv ◽  
A Genome ◽  
Target Dna ◽  
Non Homologous End Joining ◽  
Dna Strand

Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR-Cas9) system is a rapid technology for gene editing. CRISPR-Cas9 is an RNA guided gene editing tool where Cas9 acts as endonuclease by cutting the target DNA strand. Double Stranded Breaks (DBS) can be repaired by non-homologous end joining (NHEJ) and homology-directed repair (HDR). The NHEJ employs DNA ligase IV to rejoin the broken ends which cause insertion or deletion mutations, whereas HDR repairs the DSBs based on a homologous complementary template and results in perfect repair of broken ends. CRISPR-Cas9 impart diverse advantageous features in contrast with the conventional methods. In this review article, we have discussed CRISPR-Cas9 based genome editing along with its mechanism of action and role in crop improvement.

scholarly journals Erratum: Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells

2018 ◽  
Vol 36 (2) ◽  
pp. 196-196 ◽  
Author(s):  
Trung Van Chu ◽  
Timm Weber ◽  
Benedikt Wefers ◽  
Wolfgang Wurst ◽  
Sandrine Sander ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Homology Directed Repair

scholarly journals MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Linyuan Ma ◽  
Jinxue Ruan ◽  
Jun Song ◽  
Luan Wen ◽  
Dongshan Yang ◽  
...  
Keyword(s):  
Gene Editing ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Repair Capacity ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Insertion And Deletion ◽  
Large Size ◽  
Target Locus ◽  
Non Homologous End Joining

AbstractGene editing nuclease represented by Cas9 efficiently generates DNA double strand breaks at the target locus, followed by repair through either the error-prone non-homologous end joining or the homology directed repair pathways. To improve Cas9’s homology directed repair capacity, here we report the development of miCas9 by fusing a minimal motif consisting of thirty-six amino acids to spCas9. MiCas9 binds RAD51 through this fusion motif and enriches RAD51 at the target locus. In comparison to spCas9, miCas9 enhances double-stranded DNA mediated large size gene knock-in rates, systematically reduces off-target insertion and deletion events, maintains or increases single-stranded oligodeoxynucleotides mediated precise gene editing rates, and effectively reduces on-target insertion and deletion rates in knock-in applications. Furthermore, we demonstrate that this fusion motif can work as a “plug and play” module, compatible and synergistic with other Cas9 variants. MiCas9 and the minimal fusion motif may find broad applications in gene editing research and therapeutics.

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