Evaluation of site-specific homologous recombination activity of BRCA1 by direct quantitation of gene editing efficiency

AbstractThough AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, “AsCas12a Ultra”, that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

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Unsuccessful attempt at gene-editing by homologous recombination in the zebrafish germ line using the approach of “Rong and Golic”

Transgenic Research ◽

10.1007/s11248-012-9607-1 ◽

2012 ◽

Vol 21 (5) ◽

pp. 1125-1136 ◽

Cited By ~ 1

Author(s):

Rosalind Brookfield ◽

Felix Dafhnis-Calas ◽

Zhengyao Xu ◽

William Brown

Keyword(s):

Homologous Recombination ◽

Gene Editing ◽

Germ Line ◽

Unsuccessful Attempt

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One-step multiple site-specific base editing by direct embryo injection for precision and pyramid pig breeding

10.1101/2020.08.26.267948 ◽

2020 ◽

Author(s):

Ruigao Song ◽

Yu Wang ◽

Qiantao Zheng ◽

Jing Yao ◽

Chunwei Cao ◽

...

Keyword(s):

Stop Codon ◽

Point Mutations ◽

Embryonic Cells ◽

Site Specific ◽

Editing Efficiency ◽

Base Editing ◽

One Step ◽

Pig Performance ◽

Novel Alleles

AbstractPrecise and simultaneous acquisition of multiple beneficial alleles in the genome to improve pig performance are pivotal for making elite breeders. Cytidine base editors (CBEs) have emerged as powerful tools for site-specific single nucleotide replacement. Here, we compare the editing efficiency of four CBEs in porcine embryonic cells and embryos to show that hA3A-BE3-Y130F and hA3A-eBE3-Y130F consistently results in higher base-editing efficiency and lower toxic effects to in vitro embryo development. We also show that zygote microinjection of hA3A-BE3-Y130F results in one-step generation of pigs (3BE pigs) harboring C-to-T point mutations, including a stop codon in CD163 and in MSTN and induce beneficial allele in IGF2. The 3BE pigs showed improved growth performance, hip circumference, food conversion rate. Our results demonstrate that CBEs can mediate high throughput genome editing by direct embryo microinjection. Our approach allows immediate introduction of novel alleles for beneficial traits in transgene-free animals for pyramid breeding.

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Fbh1 Limits Rad51-Dependent Recombination at Blocked Replication Forks

Molecular and Cellular Biology ◽

10.1128/mcb.00471-09 ◽

2009 ◽

Vol 29 (17) ◽

pp. 4742-4756 ◽

Cited By ~ 44

Author(s):

Alexander Lorenz ◽

Fekret Osman ◽

Victoria Folkyte ◽

Sevil Sofueva ◽

Matthew C. Whitby

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Replication Fork ◽

Direct Repeat ◽

Dna Helicase ◽

Replication Forks ◽

Site Specific ◽

Replicative Stress ◽

Recombinant Formation ◽

A Site

ABSTRACT Controlling the loading of Rad51 onto DNA is important for governing when and how homologous recombination is used. Here we use a combination of genetic assays and indirect immunofluorescence to show that the F-box DNA helicase (Fbh1) functions in direct opposition to the Rad52 orthologue Rad22 to curb Rad51 loading onto DNA in fission yeast. Surprisingly, this activity is unnecessary for limiting spontaneous direct-repeat recombination. Instead it appears to play an important role in preventing recombination when replication forks are blocked and/or broken. When overexpressed, Fbh1 specifically reduces replication fork block-induced recombination, as well as the number of Rad51 nuclear foci that are induced by replicative stress. These abilities are dependent on its DNA helicase/translocase activity, suggesting that Fbh1 exerts its control on recombination by acting as a Rad51 disruptase. In accord with this, overexpression of Fbh1 also suppresses the high levels of recombinant formation and Rad51 accumulation at a site-specific replication fork barrier in a strain lacking the Rad51 disruptase Srs2. Similarly overexpression of Srs2 suppresses replication fork block-induced gene conversion events in an fbh1Δ mutant, although an inability to suppress deletion events suggests that Fbh1 has a distinct functionality, which is not readily substituted by Srs2.

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TALEN- and CRISPR-enhanced DNA homologous recombination for gene editing in zebrafish

Methods in Cell Biology - The Zebrafish - Genetics, Genomics, and Transcriptomics ◽

10.1016/bs.mcb.2016.03.005 ◽

2016 ◽

pp. 107-120 ◽

Cited By ~ 20

Author(s):

Y. Zhang ◽

H. Huang ◽

B. Zhang ◽

S. Lin

Keyword(s):

Homologous Recombination ◽

Gene Editing

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Advances in site-specific gene editing for primary immune deficiencies

Current Opinion in Allergy and Clinical Immunology ◽

10.1097/aci.0000000000000483 ◽

2018 ◽

Vol 18 (6) ◽

pp. 453-458 ◽

Cited By ~ 3

Author(s):

Caroline Y. Kuo

Keyword(s):

Gene Editing ◽

Specific Gene ◽

Site Specific ◽

Immune Deficiencies

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PML isoform expression and DNA break location relative to PML nuclear bodies impacts the efficiency of homologous recombination

Biochemistry and Cell Biology ◽

10.1139/bcb-2019-0115 ◽

2020 ◽

Vol 98 (3) ◽

pp. 314-326 ◽

Cited By ~ 2

Author(s):

Kathleen M. Attwood ◽

Jayme Salsman ◽

Dudley Chung ◽

Sabateeshan Mathavarajah ◽

Carter Van Iderstine ◽

...

Keyword(s):

Homologous Recombination ◽

Gene Editing ◽

Genotoxic Stress ◽

Nuclear Bodies ◽

Homology Directed Repair ◽

Pml Nuclear Bodies ◽

Promyelocytic Leukemia Nuclear Bodies ◽

Chromosomal Break ◽

The Impact

Promyelocytic leukemia nuclear bodies (PML NBs) are nuclear subdomains that respond to genotoxic stress by increasing in number via changes in chromatin structure. However, the role of the PML protein and PML NBs in specific mechanisms of DNA repair has not been fully characterized. Here, we have directly examined the role of PML in homologous recombination (HR) using I-SceI extrachromosomal and chromosome-based homology-directed repair (HDR) assays, and in HDR by CRISPR/Cas9-mediated gene editing. We determined that PML loss can inhibit HR in an extrachromosomal HDR assay but had less of an effect on CRISPR/Cas9-mediated chromosomal HDR. Overexpression of PML also inhibited both CRISPR HDR and I-SceI-induced HDR using a chromosomal reporter, and in an isoform-specific manner. However, the impact of PML overexpression on the chromosomal HDR reporter was dependent on the intranuclear chromosomal positioning of the reporter. Specifically, HDR at the TAP1 gene locus, which is associated with PML NBs, was reduced compared with a locus not associated with a PML NB; yet, HDR could be reduced at the non-PML NB-associated locus by PML overexpression. Thus, both loss and overexpression of PML isoforms can inhibit HDR, and proximity of a chromosomal break to a PML NB can impact HDR efficiency.

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