scholarly journals Efficient and Modular CRISPR-Cas9 Vector System for Physcomitrella patens

2019 ◽  
Author(s):  
Darren R. Mallett ◽  
Mingqin Chang ◽  
Xiaohang Cheng ◽  
Magdalena Bezanilla
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Dna Sequences ◽  
Physcomitrella Patens ◽  
Fluorescent Proteins ◽  
Stop Codon ◽  
Vector System ◽  
Null Alleles ◽  
Homology Directed Repair ◽  
Multiple Loci

ABSTRACTCRISPR-Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA-guided nuclease to initiate double-strand breaks. Until recently, classical RAD51-mediated homologous recombination has been a powerful tool for gene targeting in the moss Physcomitrella patens. However, CRISPR-Cas9 mediated genome editing in P. patens was shown to be more efficient than traditional homologous recombination (Collonnier et al. 2017). CRISPR-Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR-Cas9 genome editing in P. patens, here we describe the generation and use of a flexible and modular CRISPR-Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology-directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5’ and 3’ ends of gene coding regions. With regards to homology-directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a “stop codon cassette” with down-stream near-effortless genotyping.

scholarly journals Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

2019 ◽  
Vol 116 (42) ◽  
pp. 20959-20968 ◽  
Author(s):  
Sundaram Acharya ◽  
Arpit Mishra ◽  
Deepanjan Paul ◽  
Asgar Hussain Ansari ◽  
Mohd. Azhar ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genetic Disorders ◽  
High Specificity ◽  
Francisella Novicida ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Multiple Loci ◽  
Specificity Of Binding ◽  
Induced Pluripotent ◽  
Very High

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.

Use of Crispr/Cas Genome Editing in Ba/F3 Cells to Generate the Fip1l1-Pdgfra and Nup214-Abl1 Fusion Genes

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3575-3575
Author(s):  
Marlies Vanden Bempt ◽  
Charles E de Bock ◽  
Nicole Mentens ◽  
Olga Gielen ◽  
Ellen Geerdens ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Protein Expression ◽  
Genome Editing ◽  
Fusion Gene ◽  
Stop Codon ◽  
Chromosomal Rearrangements ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Fusion Protein Expression ◽  
A Cell

Abstract CRISPR/Cas genome editing is a powerful tool to precisely induce chromosomal breaks and to modify genes of interest. Cas9, an RNA-guided DNA endonuclease derived from Streptococcus pyogenes, is able to generate double stranded breaks (DSBs) in the genomic locus to where it is directed by its guide RNA (gRNA) component. The DSBs are subsequently repaired by one of the two main host repair mechanisms: the error-prone Non-homologous end joining (NHEJ) pathway or the very specific Homology-directed repair (HDR) pathway. We aimed to use CRISPR/Cas genome editing to generate the Fip1l1-Pdgfra and Nup214-Abl1 fusion genes by inducing chromosomal rearrangements in the interleukin-3 dependent Ba/F3 cell line. Prior to generating the chromosomal rearrangements, we optimized CRISPR/Cas genome editing in Ba/F3 cells, by targeting Cas9 to exon 24 of CD45, a cell surface transmembrane protein, of which inactivation can be easily detected by flow cytometry. Electroporation of Ba/F3 cells with plasmids expressing Cas9 and the specific guide RNA led to efficient inactivation of the CD45 gene, as measured by flow cytometry (30% of the cells showed loss of CD45 expression). The use of the Cas9 nickase variant led to an increased efficiency of CD45 inactivation with 58% of the cells showing loss of CD45 expression. We then extended these studies to assess the efficiency of homology-directed repair to introduce a specific mutation, using a single strand donor template to generate a premature stop codon in exon 24 of CD45. The successful introduction of the novel stop codon in CD45 was confirmed by PCR amplification of the targeted exon followed by massive parallel sequencing (MiSeq, Illumina) and we observed this endogenous mutation in 80% of the Ba/F3 clones. Having optimised the use and efficiency of CRISPR/Cas in Ba/F3 cells, we aimed to introduce double stranded breaks simultaneously in the genes Fip1l1 and Pdgfra to generate a cell based model for the FIP1L1-PDGFRA fusion gene as observed in chronic eosinophilic leukemia. Double strand breaks were introduced in Fip1l1 exon 23, 31, 32 or 34 together with simultaneous breaks in Pdgfra exon 12, both located on mouse chromosome 5. Upon IL3 removal, cells harbouring the deletion and fusion gene were able to survive, grow and form colonies in semi-solid medium, as was shown before for Ba/F3 cells transduced with retroviral vectors expressing FIP1L1-PDGFRA. The presence of the deletion was confirmed by PCR, and fusion protein expression was detected by Western blotting. A fusion between exon 1 of Fip1l1 and exon 12 of Pdgfra could also transform the cells, which confirmed earlier findings that the transforming capacities of the fusion protein are independent of Fip1l1 and dependent on the interruption of the juxtamembrane region of PDGFRA. The expression and phosphorylation levels of Fip1l1-Pdgfra were compared between the CRISPR/Cas generated Ba/F3 cells and retrovirally transduced cells overexpressing FIP1L1-PDGFRA. As expected, retrovirally transduced cells showed a much higher protein expression level of FIP1L1-PDGFRA, and much stronger phosphorylation compared to the CRISPR/Cas generated cells, in which the endogenous Fip1l1 promoter is used to drive the expression of the fusion protein. We also observed a difference in sensitivity to inhibition by imatinib, a kinase inhibitor with strong activity against PDGFRA. The same strategy was followed to generate a fusion between Nup214 and Abl1, as observed in a subset of T-cell acute lymphoblastic leukemia cases. Ba/F3 cells harbouring the Nup214-Abl1 fusion gene were able to survive and grow independent of IL3. The presence of the fusion gene was confirmed by PCR, and fusion protein expression was detected by Western blotting. Taken together, these data show that CRISPR/Cas induced chromosomal translocations in cells more faithfully recapitulate gene expression levels and sensitivity to chemotherapeutics when compared to retroviral transduction based expression of an oncogene. In conclusion, we have now designed and implemented an optimised platform to use CRISPR/Cas genome editing in Ba/F3 cells and measure gRNA efficacy by massive parallel sequencing. Our data confirm that the CRISPR/Cas genome editing system can be used to generate chromosomal rearrangements in Ba/F3 cells and provides a method to generate improved cell based models for the study of oncogenic tyrosine kinases. Disclosures No relevant conflicts of interest to declare.

scholarly journals Assessing the efficacy of CRISPR/Cas9 genome editing in the wheat pathogen Parastagonspora nodorum

2020 ◽  
Author(s):  
Haseena Khan ◽  
Megan C McDonald ◽  
Simon J Willams ◽  
Peter Solomon
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Pathogenic Fungus ◽  
Point Mutations ◽  
Dna Breaks ◽  
Gene Manipulation ◽  
Homology Directed Repair ◽  
Rnp Complex ◽  
Parastagonospora Nodorum

Abstract Background: The genome-editing tool CRISPR/Cas9 has revolutionized gene manipulation by providing an efficient method to generate targeted mutations. This technique deploys the Cas9 endonuclease and a guide RNA (gRNA) which interact to form a Cas9-gRNA complex that initiates gene editing through the introduction of double stranded DNA breaks. We tested the efficacy of the CRISPR/Cas9 approach as a means of facilitating a variety of reverse genetic approaches in the wheat pathogenic fungus Parastagonospora nodorum . Results: Parastagonospora nodorum protoplasts were transformed with the Cas9 protein and gRNA in the form of a preassembled ribonuclear protein (RNP) complex targeting the Tox3 effector gene. Subsequent screening of the P. nodorum transformants revealed 100% editing of those mutants screened. We further tested the efficacy of RNP complex when co-transformed with a Tox3 -Homology Directed Repair cassette harbouring 1 kb of homologous flanking DNA. Subsequent screening of resulting transformants demonstrated homologous recombination efficiencies exceeding 70%. A further transformation with a Tox3 -Homology Directed Repair cassette harbouring a selectable marker with 50 bp micro-homology flanks was also achieved 25% homologous recombination efficiency. The success of these homology directed repair approaches demonstrate that CRISPR/Cas9 is amenable to other in vivo DNA manipulation approaches such as the insertion of DNA and generating point mutations. Conclusion: These data highlight the significant potential that CRISPR/Cas9 has in expediting gene transgene-free knockouts in Parastagonospora nodorum and also in facilitating other gene manipulation approaches. Access to these tools will significantly decrease the time required to assess the requirement of gene for disease and to undertake functional studies to determine its role.

scholarly journals Fast and efficient generation of knock-in human organoids using homology-independent CRISPR/Cas9 precision genome editing

2020 ◽  
Author(s):  
Benedetta Artegiani ◽  
Delilah Hendriks ◽  
Joep Beumer ◽  
Rutger Kok ◽  
Xuan Zheng ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Mitotic Spindle ◽  
Cell Types ◽  
Intestinal Cell ◽  
Precise Integration ◽  
Exogenous Dna ◽  
Knock Out ◽  
Homology Directed Repair ◽  
Efficient Generation

AbstractCRISPR/Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences in human organoids awaits robust knock-in approaches. Here, we describe CRISPR/Cas9-mediated Homology-independent Organoid Transgenesis (CRISPR-HOT), which allows efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences at desired loci, without the necessity to inactivate TP53 in untransformed cells, previously used to increase HDR-mediated knock-in. CRISPR-HOT was employed to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter labelling the mitotic spindle by tagged tubulin and the cell membrane by tagged E-cadherin uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed TP53 involvement in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.

scholarly journals Assessing the efficacy of CRISPR/Cas9 genome editing in the wheat pathogen Parastagonspora nodorum

2020 ◽  
Author(s):  
Haseena Khan ◽  
Megan C McDonald ◽  
Simon J Willams ◽  
Peter Solomon
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Pathogenic Fungus ◽  
Point Mutations ◽  
Dna Breaks ◽  
Gene Manipulation ◽  
Homology Directed Repair ◽  
Rnp Complex ◽  
Parastagonospora Nodorum

Abstract Background: The genome-editing tool CRISPR/Cas9 has revolutionized gene manipulation by providing an efficient method to generate targeted mutations. This technique deploys the Cas9 endonuclease and a guide RNA (sgRNA) which interact to form a Cas9-sgRNA complex that initiates gene editing through the introduction of double stranded DNA breaks. We tested the efficacy of the CRISPR/Cas9 approach as a means of facilitating a variety of reverse genetic approaches in the wheat pathogenic fungus Parastagonospora nodorum. Results: Parastagonospora nodorum protoplasts were transformed with the Cas9 protein and sgRNA in the form of a preassembled ribonuclear protein (RNP) complex targeting the Tox3 effector gene. Subsequent screening of the P. nodorum transformants revealed 100% editing of those mutants screened. We further tested the efficacy of RNP complex when co-transformed with a Tox3-Homology Directed Repair cassette harbouring 1 kb of homologous flanking DNA. Subsequent screening of resulting transformants demonstrated homologous recombination efficiencies exceeding 70%. A further transformation with a Tox3-Homology Directed Repair cassette harbouring a selectable marker with 50 bp micro-homology flanks was also achieved with 25% homologous recombination efficiency. The success of these homology directed repair approaches demonstrate that CRISPR/Cas9 is amenable to other in vivo DNA manipulation approaches such as the insertion of DNA and generating point mutations. Conclusion: These data highlight the significant potential that CRISPR/Cas9 has in expediting transgene-free gene knockouts in Parastagonospora nodorum and also in facilitating other gene manipulation approaches. Access to these tools will significantly decrease the time required to assess the requirement of gene for disease and to undertake functional studies to determine its role.

scholarly journals piggyPrime: High-Efficacy Prime Editing in Human Cells Using piggyBac-Based DNA Transposition

2021 ◽  
Vol 3 ◽  
Author(s):  
Jonas Holst Wolff ◽  
Jakob Haldrup ◽  
Emil Aagaard Thomsen ◽  
Sofie Andersen ◽  
Jacob Giehm Mikkelsen
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Cell Lines ◽  
Human Cells ◽  
Vector System ◽  
High Efficacy ◽  
Genomic Alterations ◽  
Dna Transposition ◽  
Genomic Integration ◽  
Wide Range

Prime editing is a novel genome editing technology that allows a wide range of tailored genomic alterations. Prime editing does not involve homologous recombination, but suffers from low efficacy. Here, we demonstrate piggyPrime, a transfected single-vector system based on piggyBac DNA transposition for genomic integration of all prime editing components in human cells allowing easy and effective transgenesis with prime editing efficacies up to 100% in cell lines.

scholarly journals Tailor-made exopolysaccharides—CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Marius Rütering ◽  
Brady F Cress ◽  
Martin Schilling ◽  
Broder Rühmann ◽  
Mattheos A G Koffas ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Deletion ◽  
State Of The Art ◽  
Paenibacillus Polymyxa ◽  
Vector System ◽  
Wild Type ◽  
Monomer Composition ◽  
Homology Directed Repair ◽  
Highly Efficient ◽  
Micro Organisms

Abstract Application of state-of-the-art genome editing tools like CRISPR-Cas9 drastically increase the number of undomesticated micro-organisms amenable to highly efficient and rapid genetic engineering. Adaptation of these tools to new bacterial families can open up entirely new possibilities for these organisms to accelerate as biotechnologically relevant microbial factories, also making new products economically competitive. Here, we report the implementation of a CRISPR-Cas9 based vector system in Paenibacillus polymyxa, enabling fast and reliable genome editing in this host. Homology directed repair allows for highly efficient deletions of single genes and large regions as well as insertions. We used the system to investigate the yet undescribed biosynthesis machinery for exopolysaccharide (EPS) production in P. polymyxa DSM 365, enabling assignment of putative roles to several genes involved in EPS biosynthesis. Using this simple gene deletion strategy, we generated EPS variants that differ from the wild-type polymer not only in terms of monomer composition, but also in terms of their rheological behavior. The developed CRISPR-Cas9 mediated engineering approach will significantly contribute to the understanding and utilization of socially and economically relevant Paenibacillus species and extend the polymer portfolio.

scholarly journals Bayes Estimation of Species Divergence Times and Ancestral Population Sizes Using DNA Sequences From Multiple Loci

Genetics ◽  
2003 ◽  
Vol 164 (4) ◽  
pp. 1645-1656 ◽  
Author(s):  
Bruce Rannala ◽  
Ziheng Yang
Keyword(s):  
Dna Sequences ◽  
Gene Tree ◽  
Species Tree ◽  
Bayes Estimation ◽  
Ancestral Population ◽  
Divergence Times ◽  
Gene Trees ◽  
Species Divergence ◽  
Multiple Loci ◽  
Population Sizes

Abstract The effective population sizes of ancestral as well as modern species are important parameters in models of population genetics and human evolution. The commonly used method for estimating ancestral population sizes, based on counting mismatches between the species tree and the inferred gene trees, is highly biased as it ignores uncertainties in gene tree reconstruction. In this article, we develop a Bayes method for simultaneous estimation of the species divergence times and current and ancestral population sizes. The method uses DNA sequence data from multiple loci and extracts information about conflicts among gene tree topologies and coalescent times to estimate ancestral population sizes. The topology of the species tree is assumed known. A Markov chain Monte Carlo algorithm is implemented to integrate over uncertain gene trees and branch lengths (or coalescence times) at each locus as well as species divergence times. The method can handle any species tree and allows different numbers of sequences at different loci. We apply the method to published noncoding DNA sequences from the human and the great apes. There are strong correlations between posterior estimates of speciation times and ancestral population sizes. With the use of an informative prior for the human-chimpanzee divergence date, the population size of the common ancestor of the two species is estimated to be ∼20,000, with a 95% credibility interval (8000, 40,000). Our estimates, however, are affected by model assumptions as well as data quality. We suggest that reliable estimates have yet to await more data and more realistic models.

scholarly journals The M26 Hotspot of Schizosaccharomyces pombe Stimulates Meiotic Ectopic Recombination and Chromosomal Rearrangements

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1191-1204 ◽  
Author(s):  
Jeffrey B Virgin ◽  
Jeffrey P Bailey
Keyword(s):  
Homologous Recombination ◽  
Schizosaccharomyces Pombe ◽  
Dna Sequences ◽  
Chromosomal Rearrangements ◽  
Low Frequency ◽  
Repeated Sequences ◽  
Crossing Over ◽  
Ectopic Recombination ◽  
Recombination Hotspots ◽  
Integration Sites

Abstract Homologous recombination is increased during meiosis between DNA sequences at the same chromosomal position (allelic recombination) and at different chromosomal positions (ectopic recombination). Recombination hotspots are important elements in controlling meiotic allelic recombination. We have used artificially dispersed copies of the ade6 gene in Schizosaccharomyces pombe to study hotspot activity in meiotic ectopic recombination. Ectopic recombination was reduced 10–1000-fold relative to allelic recombination, and was similar to the low frequency of ectopic recombination between naturally repeated sequences in S. pombe. The M26 hotspot was active in ectopic recombination in some, but not all, integration sites, with the same pattern of activity and inactivity in ectopic and allelic recombination. Crossing over in ectopic recombination, resulting in chromosomal rearrangements, was associated with 35–60% of recombination events and was stimulated 12-fold by M26. These results suggest overlap in the mechanisms of ectopic and allelic recombination and indicate that hotspots can stimulate chromosomal rearrangements.

Single Nucleotide Substitutions Effectively Block Cas9 and Allow for Scarless Genome Editing in Caenorhabditis elegans

2021 ◽  
Author(s):  
Jeffrey C Medley ◽  
Shilpa Hebbar ◽  
Joel T Sydzyik ◽  
Anna Y. Zinovyeva
Keyword(s):  
Caenorhabditis Elegans ◽  
Genome Editing ◽  
Dna Breaks ◽  
Nucleotide Substitutions ◽  
Paternal Genome ◽  
Single Nucleotide ◽  
C Elegans ◽  
Homology Directed Repair ◽  
Maternal Genome ◽  
Guide Sequence

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize off-target sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.

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