The conversion of bacterial CRISPR/Cas defense system into a simple and efficient tool for genome manipulations brought experimental biology into new dimensions. Suddenly, genome editing reached many groups most of which were interested in it but not able to employ the available time- and labor-consuming approaches of the pre-CRISPR era. In plant biology and biotechnology, CRISPR/Cas gene editing became the second most important technology after plant transformation. Actually, it relies on the available array of methods of gene delivery. While sufficient for most purposes, the classic gene transfer methods might become a problem for some experimental settings. The main obstacle is that they include DNA delivery and, frequently, its subsequent integration into cellular genome. For this reason novel methods to achieve gene editing without the need of stable transformation and even without DNA delivery were developed. These new approaches include in vitro ribonucleoprotein complexes formulations (delivered by microinjection, particle bombardment, electroporation, liposomes etc.), use of virus-like particles and employment of bacterial secretory systems for Cas/gRNA delivery. The first attempts to achieve DNA-free editing were made less than ten years ago. Later, different types of animal and plant cells were addressed. In this mini review we try to summarize the current developments and emerging trends in the field of DNA-free editing in plants.

Pepper is the second most important vegetable crop in Bulgarian agriculture and has become the subject of extensive breeding programs that frequently employ induced mutagenesis. The success of breeding programs can be enhanced by the efficient and integral application of different biochemical and molecular methods to characterize specific mutant alleles. On the other hand, identifying new cost-effective methods is important under a limited-resources environment. In this paper we compare the levels of five health-related carotenoid compounds of fruits (α-carotene, β-carotene, lutein, β-cryptoxanthin, zeaxanthin) between a mutant variety Oranzheva kapia (possessing high ß-carotene concentration) and a corresponding initial pepper variety Pazardzhishka kapia 794. Both varieties are intended for fresh consumption. Pepper is a major natural source of β-carotene. It was observed that fruit at both commercial and botanical maturity from mutant variety had greater α-carotene and β-carotene concentrations to the initial variety (7.49 and 1.94 times higher, respectively) meaning that the mutant was superior in fruit quality to the initial genotype. Two hydroxylase enzymes, converting α- and β-carotene to lutein and zeaxanthin, respectively, are known to exist in pepper and are encoded by two genes on chromosomes 3 and 6-CrtZ_chr03 and CrtZ_chr06. The molecular characterization of the mutant variety through locus-specific Polymerase chain reaction amplification, gene cloning and sequencing as well as expression was performed. Our results suggest that the increased ß-carotene accumulation in the mutant variety Oranzheva kapia results from a biosynthetic pathway breakdown due to deletion of CrtZ_chr03 gene.

The current investigation examined the effect of inclusion of Black soldier fly (Hermetia illucens; BSF) dry larvae in hens’ diet on egg quality and hen performance. A total of 260 brown egg-laying hens (RIR × RIW) were divided into four groups (65 hens/group; 5 pens/group) and fed with control and experimental diets (inclusion rates 3%, 6%, 9%). Although the four-week feeding period did not influence production parameters, yolk color responded positively to the test diets, showing a linear increase with the percentage of BSF inclusion rate (p < 0.001). This was reaffirmed by the increased levels of yolk carotenoids (astaxanthin and β-carotene) and α-tocopherol, with notable differences in 6% and 9% BSF-fed groups (p ≤ 0.003). Lipids are an important factor in carotenoid absorption and assimilation, and the combination of fat content and carotenoids in BSF suggests the potential of this system for egg enrichment. Along with the increased antioxidant levels, a novel finding is the positive correlation between BSF inclusion rates and hens’ egg yolk levels of C:15 and C:17 fatty acids, key players in the core mechanisms of cell health and longevity. Altogether, the results provide evidence of the potential of BSF for enrichment of eggs with carotenoids and vitamins with strong antioxidant activity, which would have a positive effect on human health.