(236d) Optimizing in-Frame CRISPR/Cas9 HDR in a Plodia Interpunctella Cell Line Using a Fluorescent Reporter System for Future Creation of Rationally Designed Silk Fibroin Proteins

Utilization of naturally derived biomaterials in medical applications has been reported throughout human history. However, the current social climate has increased the interest in enhancing their properties and examining broader potential uses as a greener alternative to synthetic polymers.¹ Silk is one such material, as ancient civilizations applied spun silk fibers for sutures or wound dressings and current advancements use extracted silk fibroins (SFs) for a variety of clinical applications including drug delivery systems and tissue engineering applications.^2-4 The commercial availability of silk as a biomedical resource was facilitated by the early domestication of the silkworm, Bombyx mori, motivated not by medicinal use, but for production of fine fabrics by the textile industry. However, natural silks have evolved independently numerous times, giving rise to a vast biodiversity of silk fibers with unique protein structures and functions. Such a diversity of properties across arthropod silks could be leveraged in the biomedical field to step beyond the current B.mori derived silk fibroins into novel arenas.

Currently, the utilization of alternative silk producing species has been restricted primarily due to limited scale-up potential and limited control over rearing environments for silk production. Additionally, existing strategies for functionalizing and tuning mechanical properties of B. mori SF-based biomaterials are inefficient, expensive, or cumbersome,⁵ despite their continued dominance in the field of naturally-sourced biomaterials. Our approach is to develop alternative SF production methods for biomedical products. Recent literature and on-going international efforts have aimed to develop strategies that allow for the rational design of SFs that maintain their current advantages, but also enable protein alterations to improve functionality. Two current approaches are recombinant expression by alternative organisms or genetic modification of the silk producing organism itself, but these solutions are not without additional complications.⁶ The recombinant expression systems, especially in prokaryotes, have issues recapitulating full-length, glycosylated, highly repetitive protein structures while whole organismal genetic engineering presents challenges with efficiency and stability of transformations within B.mori silk genes. However, the expressing and processing of silk-like proteins utilizing the biological machinery that has evolved in nature, silk glands, is a superior choice, as it maintains the fiber architecture that we as humans have been unable to recapitulate in vitro. In fact, transgenic silkworms are quite capable bioreactors for high-levels of expression and efficient secretion of numerous recombinant proteins.^7-9

Herein, our objective is to advance silk biomaterials technologies and develop an efficient in-frame CRISPR/Cas9 driven homology directed repair (HDR) genetic modification strategy in a novel SF source, Plodia interpunctella, that will enable the rational design of SF-based biopolymers for applications in the biomedical field. P. interpunctella, which is a world-wide stored products insect pest, has a long history of being laboratory reared and represents an ideal candidate for substantial silk production and genetic modification capacity. Being able to rear this silk producing species indoors under controlled environments, compared to B. mori which is reared outdoors, is highly advantageous. Environmental parameters such as humidity, temperature, light cycle, diet, population dynamics, and exposure to external cues (viruses, pathogens, small molecules) as well as specific strain all impact silk quality leading to batch-to-batch variability in B. mori SF-biomaterial. Additionally, research groups from the United States have limited connections to B. mori silk farms and sericulture practices, leaving them uncertain how to bring this silkworm into the laboratory and risking accidental release of genetically modified organisms if they do not. We have established a rearing protocol that enable sufficient production of silk fibers in laboratory setting (Figure 1A). This will allow our genetic engineering work to remain under tight laboratory control, minimizing or eliminating any potential for release of a genetically modified organism into the wild.

The genome of P. interpunctella has been independently sequenced twice (accession numbers JAOPHU000000000⁴⁶ and txid58824⁴⁵⁾. A BLAST search of each genome library with a related Pyralid moth, Galleria mellonella heavy-chain fibroin partial sequence (accession # AH009792.2) resulted in the identification of sequences that had high identity to P. interpunctella Fib-H. The P. interpunctella Fib-H gene was localized to chromosome 17 in the Ag100Pests library, and an in-depth sequencing of this gene was reported in txid58824. From our bioinformatic analysis, three unique sgRNA’s were designed. Preliminary results demonstrated the feasibility of insertion of a promoter driven fluorescent marker within a P. interpunctella cell line that was transformed to constitutively express Cas9 (PiD2Cas9puro) and in the whole organism (Figure 1B). However, the efficiency in both cells and the whole organism displayed the common challenge of low integration rate (<1%) as confirmed by flow cytometry. Herein, we optimized insertion efficiency within cell line by modifying the various engineering parameters associated with CRISPR/Cas9 HDR (integration site, homology arm length, DNA donor template, delivery concentration) as assessed by flow cytometry, RT-PCR, and sequencing. Once optimized, via a promoter driven fluorescent cassette, our insertion donor was modified to remove the promoter region, switching to an in-frame strategy for the creation of chimeric SF proteins. On-going work aims to characterize the resulting gene sequences and phenotype, while future work intents are to translate this optimized strategy to the whole organism and characterize the resultant protein sequence and fiber characteristics. The development of this strategy provides a novel biomolecular tool for the rational design of silk-based biopolymers that deliver on the exceptional characteristics of native silk fibers (robust mechanical properties, high innate biocompatibility, etc.¹⁰) with additional functional peptides, that can improve this natural polymer uses in tissue engineering and drug delivery applications.

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