scholarly journals Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

2020 ◽  
Author(s):  
Lukas Villiger ◽  
Lukas Schmidheini ◽  
Nicolas Mathis ◽  
Tanja Rothgangl ◽  
Kim Marquart ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Adenosine Deaminase ◽  
Fusion Proteins ◽  
Future Directions ◽  
Site Specific ◽  
Nuclease Domain ◽  
Protospacer Adjacent Motif ◽  
Cas9 Protein ◽  
Adenine Base ◽  
Specific Nucleotide

ABSTRACTBase editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the target locus and is limited to a window within the CRISPR-Cas R-loop, where single stranded (ss)DNA is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility, and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with an adenosine deaminase we furthermore engineer adenine base editor variants (HNHx-ABE) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors, and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA.

scholarly journals Enhancing site-specific DNA integration by a Cas9 nuclease fused with a DNA donor-binding domain

2020 ◽  
Vol 48 (18) ◽  
pp. 10590-10601
Author(s):  
Shufeng Ma ◽  
Xinlong Wang ◽  
Yongfei Hu ◽  
Jie Lv ◽  
Chengfang Liu ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Sleeping Beauty ◽  
Binding Domain ◽  
Protein Fusion ◽  
Site Specific ◽  
Dna Integration ◽  
Cas9 Nuclease ◽  
Human T Cells ◽  
Dna Insertion ◽  
Aavs1 Locus

Abstract The CRISPR/Cas system is widely used for genome editing. However, robust and targeted insertion of a DNA segment remains a challenge. Here, we present a fusion nuclease (Cas9-N57) to enhance site-specific DNA integration via a fused DNA binding domain of Sleeping Beauty transposase to tether the DNA segment to the Cas9/sgRNA complex. The insertion was unidirectional and specific, and DNA fragments up to 12 kb in length were successfully integrated. As a test of the system, Cas9-N57 mediated the insertion of a CD19-specific chimeric antigen receptor (CD19-CAR) cassette into the AAVS1 locus in human T cells, and induced intrahepatic cholangiocarcinoma in mice by simultaneously mediating the insertion of oncogenic KrasG12D into the Rosa26 locus and disrupting Trp53 and Pten. Moreover, the nuclease-N57 fusion proteins based on AsCpf1 (AsCas12a) and CjCas9 exhibited similar activity. These findings demonstrate that CRISPR-associated nuclease-N57 protein fusion is a powerful tool for targeted DNA insertion and holds great potential for gene therapy applications.

scholarly journals Resources for Enhancing Alzheimer's Caregiver Health (REACH): Overview, Site-Specific Outcomes, and Future Directions

2003 ◽  
Vol 43 (4) ◽  
pp. 514-520 ◽  
Author(s):  
Richard Schulz ◽  
Louis Burgio ◽  
Robert Burns ◽  
Carl Eisdorfer ◽  
Dolores Gallagher-Thompson ◽  
...  
Keyword(s):  
Future Directions ◽  
Site Specific ◽  
Caregiver Health

Differentiation Induction In Acute Myeloid Leukemia Using Site-Specific DNA-Targeting

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3940-3940
Author(s):  
Rahul Palchaudhuri ◽  
Kwan-Keat Ang ◽  
Borja Saez ◽  
David B. Sykes ◽  
Gregory L. Verdine ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Differentiation ◽  
Calcium Binding ◽  
Fusion Proteins ◽  
Myeloid Leukemia ◽  
Dna Recognition ◽  
Control Group ◽  
Site Specific ◽  
Dna Targeting ◽  
Acute Myeloid

Abstract Hoxa9 and Meis1 are overexpressed in >70% of acute myeloid leukemia (AML) and associated with poor prognosis and survival. Hoxa9 and Meis1 interact with DNA and PBX to achieve transcription of differentiation-blocking genes. We tested transcriptional repression at Hoxa9-PBX-Meis1 genomic binding sites to induce differentiation in a model of human AML We designed a DNA-recognition strategy based on the known structure of the Hoxa9-PBX-DNA complex by fusing the DNA binding helices of Hoxa9 and PBX to create concise homeodomain fusion proteins that target the Hoxa9-PBX DNA recognition sequence. To confer transcription-repressing properties to the proteins, we attached a transcriptional repressor (sin3 interacting) domain and ectopically expressed this protein in Hoxa9-Meis1 immortalized murine progenitors. Introduction of this transcription repressor protein significantly enabled cell differentiation versus control (51.2% Mac-1high Gr-1high cells versus 11.3% for control). Multiple gene transcripts indicative of differentiation, such as GCSFR, myeloperoxidase, neutrophil elastase, and the calcium binding protein, S100A8, were also elevated in repressor-expressing cells. Furthermore, direct transcriptional targets of Hoxa9 (e.g. SOX2, CD34, FOXP1, FLT3R, DNAJC10) were down regulated in repressor-expressing cells. Importantly, a mutant repressor lacking the DNA-interacting amino acids did not affect transcription of Hoxa9 targets, demonstrating on-target specificity. Repressor-expressing cells also exhibited lower surface expression of c-Kit and Flt3 receptors and when transplanted into mice resulted in a significant increase in disease latency with a 94 day median latency versus 62 day latency for the control group (p value = 0.002). Our results demonstrate that site-specific DNA-targeting using homeodomain fusion proteins can enable AML cell differentiation and significantly increase disease latency. Disclosures: Scadden: Fate Therapeutics: Consultancy, Equity Ownership.

Three-in-One Chromatography-Free Purification, Tag Removal, and Site-Specific Modification of Recombinant Fusion Proteins Using Sortase A and Elastin-like Polypeptides

2013 ◽  
Vol 125 (13) ◽  
pp. 3791-3796 ◽  
Author(s):  
Joseph J. Bellucci ◽  
Miriam Amiram ◽  
Jayanta Bhattacharyya ◽  
Dewey McCafferty ◽  
Ashutosh Chilkoti
Keyword(s):  
Fusion Proteins ◽  
Sortase A ◽  
Site Specific ◽  
Specific Modification ◽  
Recombinant Fusion Proteins

scholarly journals Expression and regulation by interferon of a double-stranded-RNA-specific adenosine deaminase from human cells: evidence for two forms of the deaminase.

1995 ◽  
Vol 15 (10) ◽  
pp. 5376-5388 ◽  
Author(s):  
J B Patterson ◽  
C E Samuel
Keyword(s):  
Adenosine Deaminase ◽  
Fusion Proteins ◽  
Immunoblot Analysis ◽  
Binding Activity ◽  
Human Cells ◽  
Cell Fractionation ◽  
Double Stranded Rna ◽  
Nuclear Extracts ◽  
Dsrna Binding ◽  
Ifn Treatment

A 6,474-nucleotide human cDNA clone designated K88, which encodes double-stranded RNA (dsRNA)-specific adenosine deaminase, was isolated in a screen for interferon (IFN)-regulated cDNAs. Northern (RNA) blot analysis revealed that the K88 cDNA hybridized to a single major transcript of approximately 6.7 kb in human cells which was increased about fivefold by IFN treatment. Polyclonal antisera prepared against K88 cDNA products expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins recognized two proteins by Western (immunoblot) analysis. An IFN-induced 150-kDa protein and a constitutively expressed 110-kDa protein whose level was not altered by IFN treatment were detected in human amnion U and neuroblastoma SH-SY5Y cell lines. Only the 150-kDa protein was detected in mouse fibroblasts with antiserum raised against the recombinant human protein; the mouse 150-kDa protein was IFN inducible. Immunofluorescence microscopy and cell fractionation analyses showed that the 110-kDa protein was exclusively nuclear, whereas the 150-kDa protein was present in both the cytoplasm and nucleus of human cells. The amino acid sequence deduced from the K88 cDNA includes three copies of the highly conserved R motif commonly found in dsRNA-binding proteins. Both the 150-kDa and the 110-kDa proteins prepared from human nuclear extracts bound to double-stranded but not to single-stranded RNA affinity columns. Furthermore, E. coli-expressed GST-K88 fusion proteins that included the R motif possessed dsRNA-binding activity. Extracts prepared either from K88 cDNA-transfected cells or from IFN-treated cells contained increased dsRNA-specific adenosine deaminase enzyme activity. These results establish that K88 encodes an IFN-inducible dsRNA-specific adenosine deaminase and suggest that at least two forms of dsRNA-specific adenosine deaminase occur in human cells.

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