CRISPR-Based Genome Editing Tools: An Accelerator in Crop Breeding for a Changing Future

The paper addresses how CRISPR-based genome editing can accelerate crop breeding to meet food security needs under climate change. It situates CRISPR within the evolution of plant breeding from transgenics and marker-assisted selection to modern gene editing, identifying limitations in existing germplasm variation that constrain breeding gains. It introduces genome editing methods derived from natural DNA break/repair (ZFNs, TALENs) and positions CRISPR-Cas as the most versatile and widely adopted. The authors summarize CRISPR’s adaptive immunity mechanism in three stages (adaptation, crRNA biogenesis, interference), the two major classes (Class 1 multi-protein effectors vs Class 2 single-effector systems), and types I–VI, motivating a comprehensive review of CRISPR systems’ structures, PAM/PFS requirements, and applications in plant genome engineering. The purpose is to synthesize current knowledge on CRISPR tools, highlight recent advances, and discuss challenges and prospects for accelerating precise, efficient crop improvement.

This is a narrative review of CRISPR-Cas systems and their plant applications. It covers: (1) Historical genome editing approaches (ZFNs, TALENs) and their protein–DNA recognition strategies and limitations compared to CRISPR; (2) CRISPR mechanism and classification—adaptation (Cas1/2 with roles for Cas4), crRNA processing (Cas6 or RNase III/tracrRNA in Class 2), and interference guided by crRNA/tracrRNA recognizing PAM (DNA) or PFS (RNA). Two classes are outlined: Class 1 (types I, III, IV) with multi-subunit effectors (e.g., Cascade); Class 2 (types II, V, VI) with single multi-domain effectors (Cas9, Cas12, Cas13); (3) Comprehensive type-by-type survey with plant-relevant details: Type II Cas9 (HNH and RuvC cleavage, NGG PAM constraints, variants including nCas9 and dCas9 enabling base/prime editing and CRISPRa/i; examples of HDR in rice, meiotic gene edits in wheat, and RNA targeting via PAMmer); Type V Cas12a (TTTV PAM, 5′ staggered cuts, tracrRNA-independent crRNA processing enabling multiplexing; applications in rice, citrus, macroalgae; dCpf1 for transcriptional repression); Type VI Cas13 (RNA-only targeting via HEPN domains, PFS nuances; applications in RNA knockdown, plant RNA virus defense, diagnostics; dCas13 for RNA tracking and m6A editing); Class 1 Type I systems (Cas3 signature; challenges of multi-subunit delivery; Type I-E transcriptional activation in maize; Type I-D TiD with Cas10d HD nuclease hybrid features enabling small indels and long deletions up to ~7.2 kb, PAM 5′-GTH-3′); Type V-B Cas12b (small size, long sticky ends, T-rich PAMs, higher temperature optima; AaCas12b high specificity and activation potential, multiplexing, heat-tolerant editing in cotton); Type V CasΦ/Cas12j (hypercompact, minimal T-rich PAMs like 5′-TBN-3′, single RuvC for both crRNA processing and cleavage; workable in Arabidopsis/tobacco). It also reviews Type III (Cas10/CARF/HEPN modules; Cas7 RNA cleavage; diagnostics), Type IV (poorly understood, lacking some modules), and additional type V subtypes (Cas12e/CasX). The review integrates numerous plant case studies and benchmarks advantages/limitations (e.g., off-targeting, PAM restrictions, temperature sensitivity, and delivery hurdles), referencing comparative and optimization studies.

The article is a narrative, non-systematic review. The authors synthesize and interpret published primary and review literature on CRISPR-Cas systems, including structural/functional classifications, mechanistic insights, and plant-focused applications. They collate examples across crop species to illustrate tool capabilities (e.g., Cas9 HDR, Cas12a multiplexing, Cas13 antiviral defense, TiD long deletions, Cas12b thermal performance, CasΦ compact delivery), and summarize advantages, limitations, and proposed solutions (e.g., engineered PAM variants, high-fidelity nucleases, temperature-tolerant enzymes, tissue-culture-free editing, and novel delivery via nanoparticles/CPPs). No new experimental data or meta-analytic methods are presented.

- CRISPR-Cas systems offer diverse editing modalities in plants beyond Cas9, including Cas12a (Cpf1), Cas13, Cas12b, CasΦ, and Class 1 Type I systems, each with distinct PAM/PFS requirements, cut architectures, and processing mechanisms that can expand target scope and applications. - Cas9 applications: Demonstrated HDR-mediated edits in rice; creation of hexaploid wheat spo11-1 loss-of-function lines revealing meiosis functions; development of nCas9/dCas9 enabled base editors and CRISPRa/i, with applications such as drought tolerance improvement in Arabidopsis; RCas9 can target RNA with PAMmer guidance. - Cas12a advantages: T-rich PAM (TTTV), 5′ staggered cuts distal to PAM, self-processing of crRNA enabling multiplexing; in rice, STU-Cas12a multiplexed editing achieved 29.2%–50% efficiency across four targets; LbCas12a yielded higher mutation rates and longer deletions than Cas9 at EPFL9. - Cas13 (RNA editing): LwaCas13a achieved >50% knockdown for 7/9 gRNAs in rice protoplasts without collateral activity; CasRx (Cas13d) showed highest antiviral efficiency in Nicotiana benthamiana against RNA viruses with no collateral cleavage; Cas13d functions across 24–41°C, suitable for RT-RPA diagnostics; dCas13 enables programmable m6A editing by fusing writer/eraser enzymes and can map RNA–protein interactions (APEX2 fusions). Guides alone can induce Cas13-independent gene silencing (GIGS) in Arabidopsis/tobacco/tomato. - Class 1 Type I-D (TiD): Hybrid effector with Cas10d (HD nuclease) and helicase features; supports small indels and long bi-directional deletions up to ~7.2 kb in tomato; more on-target sites in tomato and Arabidopsis than Cas9; mutations heritable. - Cas12b (C2c1): Small, T-rich PAMs (VTTV), generates long sticky ends (6–8 nt), facilitating precise NHEJ outcomes; AaCas12b showed high specificity and up to 8-fold gene activation in rice; in cotton, peak editing efficiency 17.1% at 45°C for 4 days; supports multiplex and heritable edits (e.g., BvCas12b, BhCas12b v4 in Arabidopsis). - CasΦ/Cas12j: Hypercompact (~70–80 kDa), single RuvC active site for crRNA processing and DNA cleavage; minimal PAMs (e.g., 5′-TBN-3′) broaden targeting; in Arabidopsis, PDS3 edits with 8–10 bp deletions; optimization improved efficiency/specificity in Arabidopsis and tobacco. - De novo domestication: CRISPR multiplex editing rapidly domesticated crop wild relatives: Solanum pimpinellifolium lines showed ~10× more fruits, ~3× larger fruit, and ~5× lycopene vs wild type; Physalis pruinosa (groundcherry) achieved improved architecture and yield without a reference genome; allotetraploid Oryza alta edited across domestication loci toward polyploid rice crop development. - Gene stacking in polyploids: Multiplex CRISPR in wheat (Zhengmai 7698) targeted 6–15 loci via tRNA-gRNA arrays, shortening breeding from ~10 years to within ~1 year; Brassica oleracea edits for self-incompatibility and male sterility streamlined hybrid seed production; viral vectors (BNYVV, PVX) efficiently deliver multiplex gRNAs with potential transgenerational effects. - CRISPR screens: Pooled and single-cell linked CRISPR screening methods (e.g., CROP-seq, Perturb-seq) are adaptable to plants for functional genomics and phenotype discovery; imaging-coupled screens reveal regulators of lncRNA localization. - Gene drive: Highly efficient in insects (e.g., 91.4–99.6% transmission in Anopheles gambiae); in plants, limited by HDR but improved efficiency noted in Arabidopsis early egg/embryo stages; potential applications include accelerating S-gene mutant breeding, weed control, and trait improvement, pending ecological and mechanistic considerations. - Food security applications: CRISPR-generated male sterility lines in rice, wheat, soybean support hybrid seed production; edits affecting nutrient uptake (e.g., OsZIP9 for Zn, ZmbHLH121 for root aerenchyma) enhance adaptation to marginal soils; IMGE in maize enables rapid doubled haploid development within two generations. - Overcoming bottlenecks: Tissue-culture-free gene editing via de novo meristems induction; non-integrating delivery using carbon nanotubes and cell-penetrating peptides expands host range; engineering Cas variants (temperature-tolerant Cas12a/Cas12b, PAM-relaxed nucleases) and high-fidelity editors reduce off-targets and broaden applicability. - Policy/IP dimension: Complex CRISPR patent landscape and high licensing fees restrict commercial adoption; proposed solutions include focusing on alternative systems outside Cas9 IP, non-profit/free licensing models, and broader access frameworks.

The review demonstrates that expanding beyond Cas9 to include Cas12a, Cas13, Cas12b, CasΦ, and Type I systems materially broadens targetable sequence space (T-rich PAMs, minimal PAMs, RNA targets), editing outcomes (blunt vs staggered ends, long deletions), and functional applications (DNA editing, RNA knockdown, antiviral defense, epitranscriptomic modulation, transcriptional control). Such diversity addresses key constraints in plant breeding—limited variation, long breeding cycles, and complex polyploid genomes—by enabling: (i) rapid trait engineering (knockouts, base/prime edits), (ii) multiplex stacking of alleles across homoeologs, (iii) domestication of crop wild relatives for resilience traits within short timeframes, and (iv) high-throughput gene function discovery via CRISPR screens. The discussed delivery innovations (nanomaterials, CPPs) and tissue-culture-free protocols mitigate species-dependent transformation barriers, while engineered nucleases overcome PAM and temperature limitations. Collectively, these advances align with the paper’s aim to accelerate precise, efficient breeding and enhance food security under climate stress. The review also situates technological progress within regulatory and IP frameworks, recognizing that equitable licensing is pivotal for broad agricultural impact.

CRISPR-Cas tools have rapidly diversified into a versatile plant genome engineering toolbox. By detailing mechanisms, structural features, and plant-focused case studies across Cas9, Cas12a, Cas13, Cas12b, CasΦ, and Class 1 Type I systems, the review underscores how these editors enable precise, multiplex, and context-appropriate interventions that can accelerate breeding, including de novo domestication, gene stacking in polyploids, pooled functional genomics, and disease resistance. The authors highlight persistent bottlenecks—off-target risks, PAM constraints, temperature dependence, delivery and tissue culture barriers, and complex IP—that are being addressed through enzyme engineering, novel delivery platforms, tissue-culture-free approaches, and proposed licensing reforms. Future directions include: expanding temperature- and PAM-flexible editors; enhancing RNA editing and epitranscriptomic tools; improving non-integrative, species-agnostic delivery; scaling tissue-culture-free pipelines; advancing diagnostics for plant health; and integrating multiplex, multi-enzyme CRISPR systems toward synthetic plant chromosomes/genomes. These trajectories promise faster, more precise breeding of climate-resilient crops.

- Technical: Off-target mutations, PAM/PFS constraints (e.g., NGG for SpCas9, TTTV for many Cas12a), collateral RNA cleavage risk for some Cas13 variants, temperature sensitivity (Cas12a/Cas12b), and variable species- and tissue-dependence of transformation/regeneration. HDR inefficiency in many plants limits precise gene drives and knock-ins. Some systems (Type III/IV) remain complex and under-characterized for plant use. - Delivery and regeneration: Conventional plasmid/T-DNA delivery can be low-efficiency, species-dependent, and may integrate transgenes; tissue culture is labor- and time-intensive and not broadly applicable. - Maturity of tools: Emerging systems like CasΦ and several Type V subtypes require further optimization for robust, generalizable plant editing. - Regulatory/IP: Fragmented patent landscape and high licensing costs impede commercialization by smaller entities and limit broader deployment. - Review scope: As a narrative review, it does not perform a systematic or quantitative meta-analysis; coverage may be selective and not exhaustive of all plant species or CRISPR variants.