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Strategies and Protocols for Optimized Genome Editing in Potato
Thursday, 2025/02/27 | 08:28:50

Frida Meijer CarlsenIda WestbergIda Elisabeth JohansenErik AndreassonBent Larsen Petersen

CRISPR J.; 2025 Feb; 8(1):37-50. doi: 10.1089/crispr.2024.0068. 

Abstract

The potato family includes a highly diverse cultivar repertoire and has a high potential for nutritional yield improvement and refinement but must in line with other crops be adapted to biotic and abiotic stresses, for example, accelerated by climate change and environmental demands. The combination of pluripotency, high ploidy, and relative ease of protoplast isolation, transformation, and regeneration together with clonal propagation through tubers makes potato highly suitable for precise genetic engineering. Most potato varieties are tetraploid having a very high prevalence of length polymorphisms and small nucleotide polymorphisms between alleles, often complicating CRISPR-Cas editing designs and strategies. CRISPR-Cas editing in potato can be divided into (i) characterization of target area and in silico-aided editing design, (ii) isolation and editing of protoplast cells, and (iii) the subsequent explant regeneration from single protoplast cells. Implementation of efficient CRISPR-Cas editing relies on efficient editing at the protoplast (cell pool) level and on robust high-throughput editing scoring methods at the cell pool and explant level. Gene and chromatin structure are additional features to optionally consider. Strategies and solutions for addressing key steps in genome editing of potato, including light conditions and schemes for reduced exposure to hormones during explant regeneration, which is often linked to somaclonal variation, are highlighted.

 

See https://pubmed.ncbi.nlm.nih.gov/39628447/

 

FIG. 1CRISPR-Cas editing and editing scoring. Classical genome editing in plants involves a sgRNA guided Cas dsDNA nuclease with subsequent induction of the non-homologous end-joining (NHEJ) DNA repair pathway, frequently resulting in loss of function (LOF) mutations. Modulation or gain of function (MOF/GOF) may be obtained through the use of sgRNA guided base editors (BEs), for example, cytidine and adenine BEs, which employ a nickase version of the Cas enzyme (nCas) with subsequent induction of the base excision repair (BER) pathway, or by prime editor RNA (pegRNA) guided prime editors (PEs), with subsequent induction of the NHEJ or the homology directed Repair pathway (HDR), depending on cell type. While BEs have been applied successfully in plants (optimizations devised in study by Westberg et al.21 and precision reviewed in study by Molla et al.22) successful applications of PEs in plants are at present absent or scarce at best. Various mostly PCR-based editing screens exist23 of which the Indel Detection of Amplicon Analysis (IDAA) appear to be robust and very suitable for organisms with high ploidy and complex genomes, such as tetraploid potato.24,25 IDAA PCR involve a three-primer design including a fluorescently labeled, for example, fluorescein amidite (FAM), universal primer or a two-primer design with one of the gene specific forward or reverse primers being fluorescently labeled. ABE, adenine base editor; BE, base editor; CBE, cytidine base editor; IDAA, Indel Detection Amplicon Analysis; dsDNA, double stranded DNA; GOF, gain of function; Indel, Insertion/Deletion; LOF, loss of function; NHEJ, nonhomologous end-joining repair pathway; MOF, modulation of function; nCas, Nickase Cas; pDNA, plasmid DNA; PEG, polyethylene glycol; pegRNA, prime editing guide RNA; RNP, ribonucleoproten; sgRNA, sequence guide RNA; T-DNA, transfer DNA. Image created with Biorender.com.

 

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