The potential for off-target effects in CRISPR/Cas9 editing, which can be mitigated using tools like CRISPOR to predict on-target activity

The gap

Evidence profile

Sourced from the future work and inline gaps and stated challenges of the source papers, classified as general, spanning 3 journals. Those papers have been cited 2 times in total.

Supporting evidence — 3 representative gaps

• Advancing Functional Genomics in Marchantia polymorpha: A Review of Induced Mutagenesis Toolkits and Applications (2026) · Plant Molecular Biology Reporter · doi

  The diverse mutagenesis tools available for M. polymorpha each have distinct strengths and ideal applications, summa- rized in Table 1. The choice of method depends on the spe- cific research goal. For unbiased phenotype-driven screens, random mutagenesis approaches are indispensable. Heavy- ion beam irradiation currently offers the highest efficiency and diverse mutation spectrum. For testing hypotheses about specific genes, targeted editing tools like CRISPR/Cas9 are unrivalled for their speed and precision in generating knock- outs. While still developing, base and prime editing repre- sent the future for introducing specific nucleotide changes to study protein function and for trait engineering. Abbreviations: EMS, ethyl methanesulfonate; NaN ₃, sodium azide; MNU, N-methyl-N-nitrosourea; LET, linear energy transfer; DSBs, double-strand breaks; gRNA, guide RNA; NHEJ, non-homologous end joining; HDR, homol- ogy-directed repair; CRISPR, clustered regularly interspaced short palindromic repeats; TALENs, transcription activator- like effector nucleases; T-DNA, transferred DNA Several exciting frontiers lie ahead. First, the community must develop standardized, optimized protocols for random mutagenesis (e.g., EMS, heavy-ion) to build large, publicly available mutant libraries. Second, efforts to enhance HDR efficiency and adapt base/prime editing for Marchantia will unlock precise genome engineering. Third, addressing transgene expression variability through synthetic biology approaches will be crucial for complex genetic manipula- tions. Finally, integrating these mutagenesis tools with high- throughput phenotyping and sequencing technologies will enable systems-level approaches to understand gene function in this evolutionarily pivotal plant.

  generalfuture workevidence 5/5
  Keywords: mutagenesis tools approaches editing diverse available random heavy efficiency specific like crispr base prime function
• A coordinated network of ABA signaling, photorespiratory and proteostasis in EMS-induced maize mutant with enhanced drought resistance (2026) · Frontiers in Plant Science · doi

  Future research will focus on identifying the mutation site through map-based cloning or SNP analysis and validating its function via CRISPR/Cas9 gene editing. However, a limitation of the current study is that the specific causal single-nucleotide polymorphism (SNP) responsible for these pleiotropic effects has not yet been pinpointed, as EMS mutagenesis generates genome-wide variations. Additionally, further field trials under diverse environmental conditions are warranted to assess the yield stability of this mutant, bridging the gap between basic research and agronomic application.

  generalinline gapsevidence 5/5
  Keywords: future focus identifying mutation site based cloning validating function crispr gene editing limitation current speci
• CRISPR/Cas9-mediated DFR disruption suggests coordinated changes in flavonoid flux and development in Petunia × hybrida (2026) · Plant Cell Reports · cited 2× · doi

  One challenge is the potential for off-target effects in CRISPR/Cas9 editing, which can be mitigated using tools like CRISPOR to predict on-target activity. Another challenge is the need to optimize guide RNA design for efficient editing, which can be addressed using tools like CHOPCHOP. The study of DFR disruption also requires careful consideration of the complex relationships among flavonoid biosynthesis, plastidial pigments, and development in plants.

  generalstated challengesevidence 5/5
  Keywords: one challenge potential off-target effects crispr cas9 editing

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