Rapid production of SaCas9 in plant-based cell-free lysate for activity testing

Cas9 nucleases have become the most versatile tool for genome editing projects in a broad range of organisms. The recombinant production of Cas9 nuclease is desirable for in vitro activity assays or the preparation of ribonucleoproteins (RNPs) for DNA-free genome editing approaches. For the rapid production of Cas9, we explored the use of a recently established cell-free lysate from tobacco (Nicotiana tabacum L.) BY-2 cells. Using this system, the 130-kDa Cas9 nuclease from Staphylococcus aureus (SaCas9) was produced and subsequently purified via affinity chromatography. The purified apoenzyme was supplemented with ten different sgRNAs, and the nuclease activity was confirmed by the linearization of plasmid DNA containing cloned DNA target sequences.

Download Full-text

Evaluation of essential oils from 22 Guatemalan medicinal plants for in vitro activity against cancer and established cell lines

Journal of Medicinal Plants Research ◽

10.5897/jmpr2017.6528 ◽

2018 ◽

Vol 12 (3) ◽

pp. 42-49 ◽

Cited By ~ 1

Author(s):

Byron Miller Andrew ◽

Gordon Cates Rex ◽

O’Neill Kim ◽

Alfonso Fuentes Soria Juan ◽

Vicente Espinoza Luis ◽

...

Keyword(s):

Medicinal Plants ◽

Essential Oils ◽

Cell Lines ◽

Vitro Activity ◽

In Vitro Activity ◽

Established Cell Lines ◽

Established Cell

Download Full-text

A lepidopteran pacifastin member: Cloning, gene structure, recombinant production, transcript profiling and in vitro activity

Insect Biochemistry and Molecular Biology ◽

10.1016/j.ibmb.2009.03.005 ◽

2009 ◽

Vol 39 (7) ◽

pp. 430-439 ◽

Cited By ~ 13

Author(s):

Bert Breugelmans ◽

Gert Simonet ◽

Vincent van Hoef ◽

Sofie Van Soest ◽

Guy Smagghe ◽

...

Keyword(s):

Gene Structure ◽

Transcript Profiling ◽

Vitro Activity ◽

In Vitro Activity ◽

Recombinant Production

Download Full-text

PpCas9 from Pasteurella pneumotropica — a compact Type II-C Cas9 ortholog active in human cells

Nucleic Acids Research ◽

10.1093/nar/gkaa998 ◽

2020 ◽

Vol 48 (21) ◽

pp. 12297-12309

Author(s):

Iana Fedorova ◽

Aleksandra Vasileva ◽

Polina Selkova ◽

Marina Abramova ◽

Anatolii Arseniev ◽

...

Keyword(s):

Genome Editing ◽

Human Cells ◽

Type Ii ◽

Compact Type ◽

Guide Rnas ◽

Defense Systems ◽

Large Size ◽

Cas9 Nuclease ◽

Size Limitation

Abstract CRISPR-Cas defense systems opened up the field of genome editing due to the ease with which effector Cas nucleases can be programmed with guide RNAs to access desirable genomic sites. Type II-A SpCas9 from Streptococcus pyogenes was the first Cas9 nuclease used for genome editing and it remains the most popular enzyme of its class. Nevertheless, SpCas9 has some drawbacks including a relatively large size and restriction to targets flanked by an ‘NGG’ PAM sequence. The more compact Type II-C Cas9 orthologs can help to overcome the size limitation of SpCas9. Yet, only a few Type II-C nucleases were fully characterized to date. Here, we characterized two Cas9 II-C orthologs, DfCas9 from Defluviimonas sp.20V17 and PpCas9 from Pasteurella pneumotropica. Both DfCas9 and PpCas9 cleave DNA in vitro and have novel PAM requirements. Unlike DfCas9, the PpCas9 nuclease is active in human cells. This small nuclease requires an ‘NNNNRTT’ PAM orthogonal to that of SpCas9 and thus potentially can broaden the range of Cas9 applications in biomedicine and biotechnology.

Download Full-text

Synthetic CRISPR RNA-Cas9–guided genome editing in human cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1520883112 ◽

2015 ◽

Vol 112 (51) ◽

pp. E7110-E7117 ◽

Cited By ~ 82

Author(s):

Meghdad Rahdar ◽

Moira A. McMahon ◽

Thazha P. Prakash ◽

Eric E. Swayze ◽

C. Frank Bennett ◽

...

Keyword(s):

Genome Editing ◽

Gene Disruption ◽

Nuclease Activity ◽

Human Cells ◽

Chemical Modifications ◽

Guide Rna ◽

Chemically Modified ◽

System Disease ◽

Cas9 Nuclease ◽

Nuclease Digestion

Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA–RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.

Download Full-text

Introduction of Two Simultaneous Mutations By Genome Editing Greatly Enhances the Accumulation of the Endogenous Fetal Hemoglobin in Human Normal Erythroid Cells

Blood ◽

10.1182/blood.v130.suppl_1.947.947 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 947-947

Author(s):

Nikoleta Psatha ◽

Chang Li ◽

Hongjie Wang ◽

Demetri Dalas ◽

George Stamatoyannopoulos ◽

...

Keyword(s):

Genome Editing ◽

Conflicts Of Interest ◽

Fetal Hemoglobin ◽

Erythroid Differentiation ◽

Nuclease Activity ◽

Erythroid Cell ◽

Erythroid Progenitors ◽

Knock Out ◽

Gamma Globin

Abstract Thalassemia or sickle cell patients with increased fetal hemoglobin have an ameliorated clinical picture up to transfusion independency. Genome editing with targeted nucleases of the γ-globin silencer BCL11a or of the HBG promoter has been shown to reenact an HPFH-like phenotype by significantly increasing the endogenous fetal globin (HbF) expression. In order to achieve higher efficiency in HbF reactivation we developed an innovative approach, mediated through a highly customizable helper dependent-adenoviral vector (HD-Ad5/35++) with nuclease activity. This vector bares the Cas9 gene and two sequential gRNAs, permitting simultaneous targeting of two different DNA loci, specifically, the erythroid enhancer of BCL11a and the HBG promoter to recreate the -114 to -102 HPFH deletion. This double targeting vector was compared to the respective single gRNA vectors. We observed that disruption of the BCL11a-enhancer increased Gγ globin, whereas disruption of the HBG promoter had a greater impact on the Αγ globin expression. Simultaneous disruption of both loci in human erythroid progenitors increased the overall HbF expression from less than 1% in the control to more than 20%, through a possibly synergistic effect of the two mutations (HbF:2.2% in BCL11a-enhancer only knock-out, 10.8% in HBG-only knock out) by affecting both gamma globin chains. The editing efficiency per locus was similar between the single gRNA and double gRNA vectors. Erythroid cell morphology and phenotypic profile of the double knock-out cells did not differ compared to the single knock-out cells. In addition to our in vitro experiments, we observed that mobilized peripheral blood CD34+ cells transduced and edited by the double gRNA vector, can efficiently engraft in NSG mice. Furthermore, the engrafted edited cells after erythroid differentiation expressed significantly higher levels of gamma globin compared to the control. This strategy has the potential to induce higher levels of HbF reactivation with a clinical benefit in patients with beta globin disorders. Disclosures No relevant conflicts of interest to declare.

Download Full-text