scholarly journals Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells

2019 ◽  
Author(s):  
Wesley A. Wierson ◽  
Brandon W. Simone ◽  
Zachary WareJoncas ◽  
Carla Mann ◽  
Jordan M. Welker ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Human Cells ◽  
Effector Proteins ◽  
Dna Double Strand Breaks ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Strand Breaks ◽  
Guide Rnas ◽  
Protospacer Adjacent Motif ◽  
Strand Annealing

AbstractClustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) effector proteins enable the targeting of DNA double-strand breaks (DSBs) to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ~20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in Protospacer Adjacent Motif (PAM) requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale. Here, we report the first characterization of ErCas12a activity in zebrafish and human cells. Using a fluorescent reporter system, we show that CRISPR/ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR/Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR/ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR/Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.

scholarly journals Staphylococcal DNA repair is required for infection

2020 ◽  
Author(s):  
Kam Pou Ha ◽  
Rebecca S. Clarke ◽  
Gyu-Lee Kim ◽  
Jane L. Brittan ◽  
Jessica E. Rowley ◽  
...  
Keyword(s):  
Human Blood ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Reporter System ◽  
Gram Positive ◽  
Fluorescent Reporter ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Transposon Mutants

AbstractThe repair of DNA damage is essential for bacterial viability and contributes to adaptation via increased rates of mutation and recombination. However, the mechanisms by which DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double strand breaks via the oxidative burst, which are repaired by RexAB, leading to induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted survival of these pathogens in human blood, suggesting that DNA double strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection.

scholarly journals CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2518
Author(s):  
Maryam Saifaldeen ◽  
Dana E. Al-Ansari ◽  
Dindial Ramotar ◽  
Mustapha Aouida
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
Catalytic Domain ◽  
Zinc Finger Nucleases ◽  
Dna Double Strand Breaks ◽  
Transcription Activator ◽  
Strand Breaks ◽  
Protospacer Adjacent Motif ◽  
Cas9 Protein ◽  
Wide Range

The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Nevertheless, the high frequency of CRISPR system off-target activity still limits its applications, and, thus, advanced strategies for highly specific CRISPR/Cas9-mediated genome editing are continuously under development including CRISPR–FokI dead Cas9 (fdCas9). fdCas9 system is derived from linking a FokI endonuclease catalytic domain to an inactive Cas9 protein and requires a pair of guide sgRNAs that bind to the sense and antisense strands of the DNA in a protospacer adjacent motif (PAM)-out orientation, with a defined spacer sequence range around the target site. The dimerization of FokI domains generates DNA double-strand breaks, which activates the DNA repair machinery and results in genomic edit. So far, all the engineered fdCas9 variants have shown promising gene-editing activities in human cells when compared to other platforms. Herein, we review the advantages of all published variants of fdCas9 and their current applications in genome engineering.

scholarly journals Small-molecule inhibitors of histone deacetylase improve CRISPR-based adenine base editing

2021 ◽  
Author(s):  
Ha Rim Shin ◽  
Ji-Eun See ◽  
Jiyeon Kweon ◽  
Heon Seok Kim ◽  
Gi-Jun Sung ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Genome Engineering ◽  
Dna Double Strand Breaks ◽  
Reporter System ◽  
Nucleotide Substitutions ◽  
Fluorescent Reporter ◽  
Editing Efficiency ◽  
Base Editing ◽  
Adenine Base

Abstract CRISPR-based base editors (BEs) are widely used to induce nucleotide substitutions in living cells and organisms without causing the damaging DNA double-strand breaks and DNA donor templates. Cytosine BEs that induce C:G to T:A conversion and adenine BEs that induce A:T to G:C conversion have been developed. Various attempts have been made to increase the efficiency of both BEs; however, their activities need to be improved for further applications. Here, we describe a fluorescent reporter-based drug screening platform to identify novel chemicals with the goal of improving adenine base editing efficiency. The reporter system revealed that histone deacetylase inhibitors, particularly romidepsin, enhanced base editing efficiencies by up to 4.9-fold by increasing the expression levels of proteins and target accessibility. The results support the use of romidepsin as a viable option to improve base editing efficiency in biomedical research and therapeutic genome engineering.

scholarly journals Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length

2005 ◽  
Vol 25 (3) ◽  
pp. 896-906 ◽  
Author(s):  
James M. Daley ◽  
Thomas E. Wilson
Keyword(s):  
Gc Content ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Single Strand Annealing ◽  
Primary Determinant ◽  
Overhang Length ◽  
Strand Annealing

ABSTRACT The ends of spontaneously occurring double-strand breaks (DSBs) may contain various lengths of single-stranded DNA, blocking lesions, and gaps and flaps generated by end annealing. To investigate the processing of such structures, we developed an assay in which annealed oligonucleotides are ligated onto the ends of a linearized plasmid which is then transformed into Saccharomyces cerevisiae. Reconstitution of a marker occurs only when the oligonucleotides are incorporated and repair is in frame, permitting rapid analysis of complex DSB ends. Here, we created DSBs with compatible overhangs of various lengths and asked which pathways are required for their precise repair. Three mechanisms of rejoining were observed, regardless of overhang polarity: nonhomologous end joining (NHEJ), a Rad52-dependent single-strand annealing-like pathway, and a third mechanism independent of the first two mechanisms. DSBs with overhangs of less than 4 bases were mainly repaired by NHEJ. Repair became less dependent on NHEJ when the overhangs were longer or had a higher GC content. Repair of overhangs greater than 8 nucleotides was as much as 150-fold more efficient, impaired 10-fold by rad52 mutation, and highly accurate. Reducing the microhomology extent between long overhangs reduced their repair dramatically, to less than NHEJ of comparable short overhangs. These data support a model in which annealing energy is a primary determinant of the rejoining efficiency and mechanism.

Induction and Repair of DNA Double-Strand Breaks in Human Cells: Dephosphorylation of Histone H2AX and its Inhibition by Calyculin A

2005 ◽  
Vol 164 (4) ◽  
pp. 514-517 ◽  
Author(s):  
Francesca Antonelli ◽  
Mauro Belli ◽  
Giacomo Cuttone ◽  
Valentina Dini ◽  
Giuseppe Esposito ◽  
...  
Keyword(s):  
Human Cells ◽  
Double Strand Breaks ◽  
Calyculin A ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Histone H2ax

scholarly journals RNF169 limits 53BP1 deposition at DSBs to stimulate single-strand annealing repair

2018 ◽  
Vol 115 (35) ◽  
pp. E8286-E8295 ◽  
Author(s):  
Liwei An ◽  
Chao Dong ◽  
Junshi Li ◽  
Jie Chen ◽  
Jingsong Yuan ◽  
...  
Keyword(s):  
Single Strand ◽  
Fine Tuning ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Single Strand Annealing ◽  
Molecular Rheostat ◽  
Dna End Resection ◽  
Strand Annealing ◽  
End Resection

Unrestrained 53BP1 activity at DNA double-strand breaks (DSBs) hampers DNA end resection and upsets DSB repair pathway choice. RNF169 acts as a molecular rheostat to limit 53BP1 deposition at DSBs, but how this fine balance translates to DSB repair control remains undefined. In striking contrast to 53BP1, ChIP analyses of AsiSI-induced DSBs unveiled that RNF169 exhibits robust accumulation at DNA end-proximal regions and preferentially targets resected, RPA-bound DSBs. Accordingly, we found that RNF169 promotes CtIP-dependent DSB resection and favors homology-mediated DSB repair, and further showed that RNF169 dose-dependently stimulates single-strand annealing repair, in part, by alleviating the 53BP1-imposed barrier to DSB end resection. Our results highlight the interplay of RNF169 with 53BP1 in fine-tuning choice of DSB repair pathways.

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