Precise gene replacement in plants through CRISPR/Cas genome editing technology: current status and future perspectives

Abstract

CRISPR/Cas, as a simple, versatile, robust and cost-effective system for genome manipulation, has dominated the genome editing field over the past few years. The application of CRISPR/Cas in crop improvement is particularly important in the context of global climate change, as well as diverse agricultural, environmental and ecological challenges. Various CRISPR/Cas toolboxes have been developed and allow for targeted mutagenesis at specific genome loci, transcriptome regulation and epigenome editing, base editing, and precise targeted gene/allele replacement or tagging in plants. In particular, precise replacement of an existing allele with an elite allele in a commercial variety through homology-directed repair (HDR) is a holy grail in genome editing for crop improvement as it has been very difficult, laborious and time-consuming to introgress the elite alleles into commercial varieties without any linkage drag from parental lines within a few generations in crop breeding practice. However, it still remains very challenging in crop plants. This review intends to provide an informative summary of the latest development and breakthroughs in gene replacement using CRISPR/Cas technology, with a focus on achievements, potential mechanisms and future perspectives in plant biological science as well as crop improvement.

Keywords: CRISPR/Cas; Gene targeting (GT); Gene/allele replacement; Genome editing; Homology-directed repair (HDR).

Fig. 1

Two major pathways underlying the repair of DSB induced by CRISPR/Cas. There are two major pathways underlying the repair of double-stranded break (DSB) induced by CRISPR/Cas (Cas9 or Cas12a). One is a error-prone non-homologous end joining (NHEJ) pathway, which generally generates random indels for knock-out mutagenesis. Another one is homology-directed repair (HDR), which is a precise repair pathway and is generally used for targeted gene/allele replacement or knock-in

Fig. 2

Proposed Models for HDR Pathway. Three potential mechanisms are proposed for HDR in plant cells. For SSA, complementary sequences at the two ends of a DSB anneal to the other forming a chimeric DNA molecule with the 3’-overhangs trimmed. Consequently, the sequences between the complementary sequences get lost. In DSBR, following the DSB induction, free 3’ overhangs are formed via exonuclease resection. 3’ ends interact with their homologous sequences in the donor repair templates, which leads to the formation of a Holliday junction. Resolution of the junction results in two DNA molecules that either have cross-over or gene conversion. In SDSA, the DSB is firstly resected and processed to generate 3’ overhangs on both sides of the DSB. The 3’ overhangs are then paired with the homologous arms of the donor repair template (DRT) and are extended by DNA synthesis. Finally, newly synthesized strands withdraw from the donor templates and anneal back to the locus. In addition, repair templates switch between donors and wild type during DNA synthesis, usually results in the partial HDR events. When donor fragment is dsDNA, either strand of the dsDNA can act as repair template. DSB: double strand break, SSA: single-strand annealing, DSBR: so-called double-strand break repair, SDSA: synthesis-dependent strand annealing, LHA: left homology arm, RHA: right homology arm, RT: Reverse transcription. ⋆:Nucleotides changes in DRT