scholarly journals CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks

2020 ◽  
Author(s):  
Joshua C. Cofsky ◽  
Deepti Karandur ◽  
Carolyn J. Huang ◽  
Isaac P. Witte ◽  
John Kuriyan ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Strand Break ◽  
Loop Structure ◽  
Loop Formation ◽  
Strand Breaks ◽  
Guide Rna ◽  
Double Stranded Dna ◽  
Dna Strands ◽  
Type V

ABSTRACTMost type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing applications. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one of the DNA strands of a double-helical substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we show that Cas12a-mediated R-loop formation destabilizes DNA at the second-strand cleavage site, which is located outside of the R-loop structure and beyond the 3′ end of the guide RNA. Chemical and fluorescent DNA probes reveal that this destabilization is an intrinsic feature of DNA flanking the RNA-3′ side of R-loops and does not require direct protein interactions. Interestingly, DNA flanking the RNA-5′ side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

scholarly journals CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Joshua C Cofsky ◽  
Deepti Karandur ◽  
Carolyn J Huang ◽  
Isaac P Witte ◽  
John Kuriyan ◽  
...  
Keyword(s):  
Genome Editing ◽  
Active Site ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Loop Structure ◽  
Strand Breaks ◽  
Guide Rna ◽  
Double Stranded Dna ◽  
Type V ◽  
Dna Substrate

Type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one strand of a double-helical DNA substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we detect intrinsic instability in DNA flanking the RNA-3′ side of R-loops, which Cas12a can exploit to expose second-strand DNA for cutting. Interestingly, DNA flanking the RNA-5′ side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

scholarly journals CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

2020 ◽  
Author(s):  
Anindya Bandyopadhyay ◽  
Nagesh Kancharla ◽  
vivek javalkote ◽  
santanu dasgupta ◽  
Thomas Brutnell
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Temperature Stability ◽  
Double Strand Breaks ◽  
Plant Genome ◽  
Strand Breaks ◽  
Guide Rna ◽  
Versatile Tool ◽  
Type V ◽  
Global Population

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA (tracrRNA), targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

scholarly journals Beyond reversal: ubiquitin and ubiquitin-like proteases and the orchestration of the DNA double strand break repair response

2019 ◽  
Vol 47 (6) ◽  
pp. 1881-1893
Author(s):  
Alexander J. Garvin
Keyword(s):  
Protein Interactions ◽  
Cellular Response ◽  
Strand Break ◽  
Central Component ◽  
Protein Protein Interactions ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Post Translational Modifications ◽  
Strand Breaks ◽  
Dna Double Strand Break

The cellular response to genotoxic DNA double strand breaks (DSBs) uses a multitude of post-translational modifications to localise, modulate and ultimately clear DNA repair factors in a timely and accurate manner. Ubiquitination is well established as vital to the DSB response, with a carefully co-ordinated pathway of histone ubiquitination events being a central component of DSB signalling. Other ubiquitin-like modifiers (Ubl) including SUMO and NEDD8 have since been identified as playing important roles in DSB repair. In the last five years ∼20 additional Ub/Ubl proteases have been implicated in the DSB response. The number of proteases identified highlights the complexity of the Ub/Ubl signal present at DSBs. Ub/Ubl proteases regulate turnover, activity and protein–protein interactions of DSB repair factors both catalytically and non-catalytically. This not only ensures efficient repair of breaks but has a role in channelling repair into the correct DSB repair sub-pathways. Ultimately Ub/Ubl proteases have essential roles in maintaining genomic stability. Given that deficiencies in many Ub/Ubl proteases promotes sensitivity to DNA damaging chemotherapies, they could be attractive targets for cancer treatment.

scholarly journals Human Gene Targeting by Adeno-Associated Virus Vectors Is Enhanced by DNA Double-Strand Breaks

2003 ◽  
Vol 23 (10) ◽  
pp. 3550-3557 ◽  
Author(s):  
Daniel G. Miller ◽  
Lisa M. Petek ◽  
David W. Russell
Keyword(s):  
Gene Targeting ◽  
Human Gene ◽  
Retroviral Vectors ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Adeno Associated Virus ◽  
Strand Breaks ◽  
Double Stranded Dna ◽  
Linear Molecules

ABSTRACT The use of adeno-associated virus (AAV) to package gene-targeting vectors as single-stranded linear molecules has led to significant improvements in mammalian gene-targeting frequencies. However, the molecular basis for the high targeting frequencies obtained is poorly understood, and there could be important mechanistic differences between AAV-mediated gene targeting and conventional gene targeting with transfected double-stranded DNA constructs. Conventional gene targeting is thought to occur by the double-strand break (DSB) model of homologous recombination, as this can explain the higher targeting frequencies observed when DSBs are present in the targeting construct or target locus. Here we compare AAV-mediated gene-targeting frequencies in the presence and absence of induced target site DSBs. Retroviral vectors were used to introduce a mutant lacZ gene containing an I-SceI cleavage site and to efficiently deliver the I-SceI endonuclease, allowing us to carry out these studies with normal and transformed human cells. Creation of DSBs by I-SceI increased AAV-mediated gene-targeting frequencies 60- to 100-fold and resulted in a precise correction of the mutant lacZ reporter gene. These experiments demonstrate that AAV-mediated gene targeting can result in repair of a DNA DSB and that this form of gene targeting exhibits fundamental similarities to conventional gene targeting. In addition, our findings suggest that the selective creation of DSBs by using viral delivery systems can increase gene-targeting frequencies in scientific and therapeutic applications.

scholarly journals Reduced DNA repair during differentiation of a myogenic cell line.

1976 ◽  
Vol 70 (3) ◽  
pp. 685-691 ◽  
Author(s):  
A C Chan ◽  
I G Walker
Keyword(s):  
Strand Break ◽  
Post Treatment ◽  
Single Strand ◽  
Repair Synthesis ◽  
Single Strand Break ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Before And After ◽  
Dna Strands ◽  
A Minor

Repair synthesis induced by 4-nitroquinoline-1-oxide (4NQO) in L6 myoblasts before and after cellular fusion was measured by [3H] thymidine incorporation into unreplicated DNA. The level of repair synthesis was reuced after the cells had fused into myotubes. The terminal addition of radioactive nucleotides into DNA strands occurred only to a minor extent, and the dilution of [3H] thymidine by intracellular nucleotide pools was shown not to be responsible for the observed difference in repair synthesis, Both the initial rate and the overall incorporation of [3H] thymidine were found to be 50% lower in the myotubes. 4NQO treatment of myoblasts and myotubes induced modifications in the DNA which were observed as single-strand breaks during alkaline sucrose sedimentation. After the myoblasts were allowed a post-treatment incubation, most of the single-strand breaks were not longer apparent. In contrast, a post-treatment incubation of myotubes did not change the extent of single-strand breakage seen. Both myoblasts and myotubes were equally effective in repairing single-strand breaks induced by X radiation. It would appear that when myoblasts fuse, a repair enzyme activity is lost, probably an endonuclease that recognizes one of the 4 NQO modifications of DNA. The result observed is a partial loss of repair synthetic ability and a complete loss of ability to remove the modification that appears as a single-strand break in alkali.

scholarly journals Polymerase theta-helicase promotes end joining by stripping single-stranded DNA-binding proteins and bridging DNA ends

2021 ◽  
Author(s):  
Jeffrey M Schaub ◽  
Michael M Soniat ◽  
Ilya J Finkelstein
Keyword(s):  
Single Molecule ◽  
Protein A ◽  
Strand Break ◽  
Helicase Domain ◽  
End Joining ◽  
Single Stranded Dna ◽  
Double Stranded Dna ◽  
Dna Strands ◽  
In Trans

Homologous recombination-deficient cancers rely on DNA polymerase Theta (Polθ)-Mediated End Joining (TMEJ), an alternative double-strand break repair pathway. Polθ is the only vertebrate polymerase that encodes an N-terminal superfamily 2 (SF2) helicase domain, but the role of this helicase domain in TMEJ remains unclear. Using single-molecule imaging, we demonstrate that Polθ-helicase (Polθ-h) is a highly processive single-stranded DNA (ssDNA) motor protein that can efficiently strip Replication Protein A (RPA) from ssDNA. Polθ-h also has a limited capacity for disassembling RAD51 filaments but is not processive on double- stranded DNA. Polθ-h can bridge two non-complementary DNA strands in trans. PARylation of Polθ-h by PARP-1 resolves these DNA bridges. We conclude that Polθ-h removes RPA and RAD51 filaments and mediates bridging of DNA overhangs to aid in polymerization by the Polθ polymerase domain.

Protein–protein interactions during mammalian DNA single-strand break repair

2003 ◽  
Vol 31 (1) ◽  
pp. 247-251 ◽  
Author(s):  
K.W. Caldecott
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Single Strand ◽  
Protein Protein Interactions ◽  
Single Strand Break ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Single Strand Break Repair

The genetic stability of living cells is continually threatened by endogenous reactive oxygen species and other genotoxic molecules. Of particular threat are the thousands of single-strand breaks that arise in each cell every day. If left unrepaired, such breaks can give rise to potentially clastogenic or lethal chromosomal double-strand breaks. This article summarizes our current understanding of how mammalian cells detect and repair single strand breaks, and provides insights into novel polypeptide components of this process.

scholarly journals Topological DNA-binding of structural maintenance of chromosomes-like RecN promotes DNA double-strand break repair in Escherichia coli

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Kenji Keyamura ◽  
Takashi Hishida
Keyword(s):  
Escherichia Coli ◽  
Strand Break ◽  
Dependent Manner ◽  
Loop Formation ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
D Loop ◽  
Linear Dsdna ◽  
Structural Maintenance Of Chromosomes

AbstractBacterial RecN, closely related to the structural maintenance of chromosomes (SMC) family of proteins, functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination. Here we show that the purified Escherichia coli RecN protein topologically loads onto both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) that has a preference for ssDNA. RecN topologically bound to dsDNA slides off the end of linear dsDNA, but this is prevented by RecA nucleoprotein filaments on ssDNA, thereby allowing RecN to translocate to DSBs. Furthermore, we found that, once RecN is recruited onto ssDNA, it can topologically capture a second dsDNA substrate in an ATP-dependent manner, suggesting a role in synapsis. Indeed, RecN stimulates RecA-mediated D-loop formation and subsequent strand exchange activities. Our findings provide mechanistic insights into the recruitment of RecN to DSBs and sister chromatid interactions by RecN, both of which function in RecA-mediated DSB repair.

scholarly journals CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles

2018 ◽  
Vol 115 (9) ◽  
pp. E2040-E2047 ◽  
Author(s):  
Brenda R. Lemos ◽  
Adam C. Kaplan ◽  
Ji Eun Bae ◽  
Alexander E. Ferrazzoli ◽  
James Kuo ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Mammalian Cells ◽  
Budding Yeast ◽  
Nuclease Activity ◽  
Strand Break ◽  
End Joining ◽  
Guide Rna ◽  
Dna Double Strand Break ◽  
Nhej Repair ◽  
Dna Strands

Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5′ overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9–mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11’s nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

scholarly journals Type II and type V CRISPR effector nucleases from a structural biologist’s perspective

2016 ◽  
Vol 62 (3) ◽  
pp. 315-326
Author(s):  
Humberto Fernandes ◽  
Michal Pastor ◽  
Matthias Bochtler
Keyword(s):  
Target Sequence ◽  
Type Ii ◽  
Molecular Architecture ◽  
Genomic Engineering ◽  
Double Stranded Dna ◽  
Target Strand ◽  
Target Dna ◽  
Dna Strands ◽  
Catalytic Role ◽  
Type V

The type II and type V CRISPR effector nucleases Cas9 and Cpf1 are “universal” DNA endonucleases, which can be programmed by an appropriate crRNA or sgRNA strand to cleave almost any DNA duplex at a preselected position (constrained only by short, so-called PAMs). In this review, we briefly introduce CRISPR bacterial adaptive immunity as the biological context in which Cas9 and Cpf1 proteins operate, and then present the structural insights that have been obtained in the last two or three years that illustrate the mode of operation of these proteins. We describe the R-loop structures at the core of the Cas9 and Cpf1 complexes, and the structure of the 5’- or 3’-handles that help anchor the nucleic acid complexes to the proteins in a manner that is independent of the target sequence. Next, we describe the molecular architecture of the Cas9 and Cpf1 proteins. We illustrate how Cas9 and Cpf1 proteins scan double stranded DNA for so-called protospacer associated motifs (PAMs), we explain how the phosphate loop (PLL) and basic helix (BH) promote the separation of target and non-target DNA strands and the formation of hybrids between crRNA or sgRNA and the target strand of DNA. We also describe the current understanding of the catalytic mechanisms of RuvC and HNH domains, and a possible, but still very uncertain catalytic role of the Nuc domain. At the end of the review, we briefly summarize key developments that have initiated the field of genomic engineering using Cas9 or Cpf1 nucleases..

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