Identifying phenotypes of silk altered by CRISPR/Cas9 gene editing of fibroin light chain gene in the Indian meal moth (Plodia interpunctella)

Natural silk is produced across most of the 160,000 lepidopteran species as a versatile, multifunctional biopolymer that is produced during a variety of physiological events within the taxa. Historically, silk from the domesticated silkworm, Bombyx mori, has been used for many human applications including clothing, furniture, medicine, and biomaterials and consequently has received the most research. Unfortunately, this singular focus on silkworm silk has created a knowledge gap where little is known about the mechanical properties of natural silk or the genetic mechanisms underlying silk production in the diverse array of other butterfly and moth species. To broaden the study of genetic to phenotypic relationships, the Indian meal moth, Plodia interpunctella, was selected as an alternative model organism. Using CRISPR/Cas9 mediated gene editing, the fibroin light chain (PiFibL) will be mutagenized, creating Non-Homologous End Joining indels at PAM sites within the coding sequence. Using transcript sequence for PiFibL, two potential CRISPR-PAM sites were identified within exon 3 and exon 7using ZiFit 4.2. The phenotype for PiFibL knockouts was assayed by isolating larvae of CRISPR/Cas9 microinjected embryos into individual rearing containers and visualizing quantity and quality of silk produced. Three unique phenotypes were observed: 1) reduced larval silking, 2) no/little pupal cocoon silk, and 3) patchy silk sheeting. However, genomic sequencing of individuals did not identify indel mutations at the PAM sites. Future studies will focus on tagging and identifying PiFibL knockout phenotypes and improving genomic sequencing of the locus to improve PAM site sequencing.