scholarly journals Precision genome editing in plants via gene targeting and subsequent break‐induced single‐strand annealing

2020 ◽  
Author(s):  
Masaki Endo ◽  
Satoshi Iwakami ◽  
Seiichi Toki
Keyword(s):  
Genome Editing ◽  
Gene Targeting ◽  
Single Strand ◽  
Single Strand Annealing ◽  
Strand Annealing

scholarly journals Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants

2021 ◽  
Vol 2 ◽  
Author(s):  
Namie Ohtsuki ◽  
Keiko Kizawa ◽  
Akiko Mori ◽  
Ayako Nishizawa-Yokoi ◽  
Takao Komatsuda ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Targeting ◽  
Mirna Target ◽  
Single Strand ◽  
Target Site ◽  
Selection Marker ◽  
Base Substitution ◽  
Marker Excision ◽  
Single Strand Annealing ◽  
Strand Annealing

Gene targeting (GT) enables precise genome modification—e.g., the introduction of base substitutions—using donor DNA as a template. Combined with clean excision of the selection marker used to select GT cells, GT is expected to become a standard, generally applicable, base editing system. Previously, we demonstrated marker excision via a piggyBac transposon from GT-modified loci in rice. However, piggyBac-mediated marker excision has the limitation that it recognizes only the sequence TTAA. Recently, we proposed a novel and universal precise genome editing system consisting of GT with subsequent single-strand annealing (SSA)-mediated marker excision, which has, in principle, no limitation of target sequences. In this study, we introduced base substitutions into the microRNA miR172 target site of the OsCly1 gene—an ortholog of the barley Cleistogamy1 gene involved in cleistogamous flowering. To ensure efficient SSA, the GT vector harbors 1.2-kb overlapped sequences at both ends of a selection marker. The frequency of positive–negative selection-mediated GT using the vector with overlapped sequences was comparable with that achieved using vectors for piggyBac-mediated marker excision without overlapped sequences, with the frequency of SSA-mediated marker excision calculated as ~40% in the T0 generation. This frequency is thought to be adequate to produce marker-free cells, although it is lower than that achieved with piggyBac-mediated marker excision, which approaches 100%. To date, introduction of precise substitutions in discontinuous multiple bases of a targeted gene using base editors and the prime editing system based on CRISPR/Cas9 has been quite difficult. Here, using GT and our SSA-mediated marker excision system, we succeeded in the precise base substitution not only of single bases but also of artificial discontinuous multiple bases in the miR172 target site of the OsCly1 gene. Precise base substitution of miRNA target sites in target genes using this precise genome editing system will be a powerful tool in the production of valuable crops with improved traits.

scholarly journals Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in Leishmania

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Wen-Wei Zhang ◽  
Greg Matlashewski
Keyword(s):  
Gene Targeting ◽  
Mammalian Cells ◽  
Gene Editing ◽  
Single Strand ◽  
End Joining ◽  
Content Type ◽  
Single Strand Annealing ◽  
Dna Break ◽  
Strand Annealing ◽  
Double Strand Dna

ABSTRACT CRISPR-Cas9 genome editing relies on an efficient double-strand DNA break (DSB) and repair. Contrary to mammalian cells, the protozoan parasite Leishmania lacks the most efficient nonhomologous end-joining pathway and uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair to repair DSBs. Here, we reveal that Leishmania predominantly uses single-strand annealing (SSA) (>90%) instead of MMEJ (<10%) for DSB repair (DSBR) following CRISPR targeting of the miltefosine transporter gene, resulting in 9-, 18-, 20-, and 29-kb sequence deletions and multiple gene codeletions. Strikingly, when targeting the Leishmania donovani LdBPK_241510 gene, SSA even occurred by using direct repeats 77 kb apart, resulting in the codeletion of 15 Leishmania genes, though with a reduced frequency. These data strongly indicate that DSBR is not efficient in Leishmania, which explains why more than half of DSBs led to cell death and why the CRISPR gene-targeting efficiency is low compared with that in other organisms. Since direct repeat sequences are widely distributed in the Leishmania genome, we predict that many DSBs created by CRISPR are repaired by SSA. It is also revealed that DNA polymerase theta is involved in both MMEJ and SSA in Leishmania. Collectively, this study establishes that DSBR mechanisms and their competence in an organism play an important role in determining the outcome and efficacy of CRISPR gene targeting. These observations emphasize the use of donor DNA templates to improve gene editing specificity and efficiency in Leishmania. In addition, we developed a novel Staphylococcus aureus Cas9 constitutive expression vector (pLdSaCN) for gene targeting in Leishmania. IMPORTANCE Due to differences in double-strand DNA break (DSB) repair mechanisms, CRISPR-Cas9 gene editing efficiency can vary greatly in different organisms. In contrast to mammalian cells, the protozoan parasite Leishmania uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair (HDR) to repair DSBs but lacks the nonhomologous end-joining pathway. Here, we show that Leishmania predominantly uses single-strand annealing (SSA) instead of MMEJ for DSB repairs (DSBR), resulting in large deletions that can include multiple genes. This strongly indicates that the overall DSBR in Leishmania is inefficient and therefore can influence the outcome of CRISPR-Cas9 gene editing, highlighting the importance of using a donor DNA to improve gene editing fidelity and efficiency in Leishmania.

scholarly journals The Yeast Recombinational Repair Protein Rad59 Interacts With Rad52 and Stimulates Single-Strand Annealing

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 515-525 ◽  
Author(s):  
Allison P Davis ◽  
Lorraine S Symington
Keyword(s):  
Protein A ◽  
Single Strand ◽  
Physical Interaction ◽  
Dna Double Strand Breaks ◽  
Single Stranded Dna ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Recombination Pathway

Abstract The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.

scholarly journals Saw1 localizes to repair sites but is not required for recruitment of Rad10 to repair intermediates bearing short non-homologous 3′ flaps during single-strand annealing in S. cerevisiae

2015 ◽  
Vol 412 (1-2) ◽  
pp. 131-139
Author(s):  
Melina Mardirosian ◽  
Linette Nalbandyan ◽  
Aaron D. Miller ◽  
Claire Phan ◽  
Eric P. Kelson ◽  
...  
Keyword(s):  
Single Strand ◽  
Single Strand Annealing ◽  
Strand Annealing

Gene cloning and seamless site-directed mutagenesis using single-strand annealing (SSA)

2018 ◽  
Vol 102 (23) ◽  
pp. 10119-10126
Author(s):  
Zhixin Luo ◽  
Shanhe Wang ◽  
Beilei Jiao ◽  
Dan Yuan ◽  
Dongmei Dai ◽  
...  
Keyword(s):  
Gene Cloning ◽  
Single Strand ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Single Strand Annealing ◽  
Strand Annealing

scholarly journals Human RECQL4 represses the RAD52‐mediated single‐strand annealing pathway after ionizing radiation or cisplatin treatment

2020 ◽  
Vol 146 (11) ◽  
pp. 3098-3113 ◽  
Author(s):  
Masaoki Kohzaki ◽  
Akira Ootsuyama ◽  
Lue Sun ◽  
Takashi Moritake ◽  
Ryuji Okazaki
Keyword(s):  
Ionizing Radiation ◽  
Single Strand ◽  
Single Strand Annealing ◽  
Strand Annealing
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