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Review
, 12 (1), 95

Challenges and Perspectives in Homology-Directed Gene Targeting in Monocot Plants

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Review

Challenges and Perspectives in Homology-Directed Gene Targeting in Monocot Plants

Tien Van Vu et al. Rice (N Y).

Abstract

Continuing crop domestication/redomestication and modification is a key determinant of the adaptation and fulfillment of the food requirements of an exploding global population under increasingly challenging conditions such as climate change and the reduction in arable lands. Monocotyledonous crops are not only responsible for approximately 70% of total global crop production, indicating their important roles in human life, but also the first crops to be challenged with the abovementioned hurdles; hence, monocot crops should be the first to be engineered and/or de novo domesticated/redomesticated. A long time has passed since the first green revolution; the world is again facing the challenge of feeding a predicted 9.7 billion people in 2050, since the decline in world hunger was reversed in 2015. One of the major lessons learned from the first green revolution is the importance of novel and advanced trait-carrying crop varieties that are ideally adapted to new agricultural practices. New plant breeding techniques (NPBTs), such as genome editing, could help us succeed in this mission to create novel and advanced crops. Considering the importance of NPBTs in crop genetic improvement, we attempt to summarize and discuss the latest progress with major approaches, such as site-directed mutagenesis using molecular scissors, base editors and especially homology-directed gene targeting (HGT), a very challenging but potentially highly precise genome modification approach in plants. We therefore suggest potential approaches for the improvement of practical HGT, focusing on monocots, and discuss a potential approach for the regulation of genome-edited products.

Keywords: CRISPR/Cas; Gene targeting (GT); Homology-directed gene targeting (HGT); Homology-directed repair (HDR); Monocots; Precision breeding; Targeted mutagenesis.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
DSB repair pathways.1017In the C-NHEJ pathway, DSB formation induces binding to broken ends by KU70/80 heterodimers that subsequently recruit the DNA damage response kinase (DDK) complex such as DNA-PKcs in mammals. DDK then activates the 53BP1/RIF1 complex, which plays a role in shielding the broken ends from resection by antagonizing BRCA1/CtIP activity. DNA-PK also activates other KU-recruited proteins, such as XLF, XRCC4 and Lig4, for ligating the broken ends. In the HDR pathway, DSB formation induces cell cycle arrest initiated with the activation of ATM resulting from sensing a chromatin structure change. Activated monomeric ATM then phosphorylates the MRN complex and P53/SOG1, which regulates the cell cycle checkpoint and arrest. MRN activation supports end resection for HDR. Limited resection leads to MMEJ, and if a substantial level of resection is formed in the absence of a donor template, SSA is likely to be used for the repair. MMEJ requires PARP and Pol Q for its processes, and SSA requires the role of RAD52. Both MMEJ and SSA require the ssDNA flap endonuclease FEN1 and Lig3/Lig1:XRCC1 for ligating final products. Extensive resection of the broken ends is facilitated by Exonuclease 1 (Exo1) and/or Dna2. In the presence of donor template, the 3′ overhangs of resected ends could be protected by RPA binding and then recruiting RAD51 to the ssDNA with support and control by BRCA2. RAD51 binds to the resected ssDNA overhang, forming nucleoprotein filaments or presynaptic filaments. With the support of RAD54, the filament structure invades the donor template sequence and searches for and anneals to the complementary sequence; then, displacement loop (D-loop) formation occurs. Subsequently, the free 3′ OH end of the invaded ssDNA primes donor template-dependent DNA synthesis. This process determines the outcomes of HDR with several sub-pathways (DSBR with dHJ and SDSA) with the supportive activity of RAD5A, RECQ4A and MUS81. The DNA fragments and protein structures are not pictured to scale. The potential proteins involved in the processes of each pathway or sub-pathway are denoted adjacent to their approaching lines. XRCC: X-ray repair cross-complementing protein; XLF: XRCC4-like factor; Lig4: DNA ligase 4; PARP: poly-ADP-ribose polymerase; Pol Q: DNA polymerase theta
Fig. 2
Fig. 2
Four generations of molecular scissors. The first, second and third generations of molecular scissors, Homing nuclease (a); ZFN (b); and TALEN (c), are characterized as nucleases relying on DNA binding domains to recognize DNA target sites. Homing nucleases recognize long DNA sequences of 14–40 bp with their DNA binding domains. A ZFN or TALEN is designed by connecting 3–6 zinc finger motifs or 17–20 TALE modules, respectively, for DNA binding and an endonuclease domain of FokI restriction enzyme for cutting. FokI works only in homodimer form, so usually one has to design pairs of ZFNs or TALENs to target a DNA site. FokI activity usually produces DSB with 4 nt overhangs. The fourth generation, CRISPR/Cas (d), is also the most powerful one; it uses guide RNA components to form active complexes, thereby interrogating and searching for target DNA sites based on Watson-Crick base pairing between the guide RNA and targeted strand. The DNA fragments and protein structures are not pictured to scale
Fig. 3
Fig. 3
Non-DSB precise gene targeting approaches. a Base of approach editing. Cytosine Base Editors (CBEs) and Adenosine Base Editors (ABEs) are the two types of base editors that have been published so far. CBEs: Dead Cas9 (blue) binds to target C (green) via the RNA (pink) guide, which mediates the separation of local DNA strands. A tethered APOBEC1 (green) enzyme by cytosine deamination converts the single-stranded target C to U. The initial G: C is replaced by the A: T base pair at the target location through DNA repair or replication. ABEs: A hypothetical deoxyadenosine deaminase (red) and catalytically impaired nCas9 (Cas9 D10A nickase) bind target DNA in the RNA guide to expose a small bubble of single-stranded DNA that catalyzes the conversion of A to I within this bubble. b Oligonucleotide-directed mutagenesis process. A gene repair oligonucleotide (GRON), which contains designed modifications, is delivered and paired with the target DNA sequence. GRON creates a mismatch at the target site and triggers a DNA repair mechanism. DNA repair enzymes detect the mismatch and repair the target DNA sequence using GRON as a template. Once the repair process is completed during cell division and multiplication, the GRON is removed and degraded. The target sequence is modified with designed changes. The representative DNA fragments and protein structures are not pictured to scale. c Prime editing. Prime editor is a CRISPR/Cas complex developed by fusion of a reverse transcriptase (RT) to a C-terminal of nickase Cas9 (H840A) and a prime editing gRNA (pegRNA) with a 3 ‘extension that could bind to the 3 ‘nicked strands produced by the nCas9. When bound, the 3′-OH free nicked strand is used as a substratum for the RT to copy genetic information from the 3 ‘extension of pegRNA
Fig 4
Fig 4
Homology-directed repair pathway determination and its favorable cell contexts. Activation of the MRN complex and P53/SOG1 triggers the activation of cell cycle checkpoint proteins such as CDKB1 (G2/M checkpoint) and CYCB1 (S phase checkpoint) or NAC-type transcription factors ANAC044 and ANAC085 (S/G2 checkpoints) or WEE1 kinase for cell cycle arrest

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