Part:BBa_K5167669

CRISPR/Cas9 target site sequence of OsGluB-1

This is a target site sequence for crispr/cas9 recognition in a protein coding gene that encodes the glutelin B1 in rice endosperm proteins. It is located on the 8458664-8460633 reverse strand and the 8465066-8467035 forward strand of Chromosome 2:. Its length is 1650 bases, including 4 exon regions. When it mutates simultaneously with Osglb, it can significantly increase the total protein, prolamine, and lysine content in rice. Utilize its mutation and dose compensation effect expressed between rice storage proteins to increase total protein, prolamine, and lysine content in rice, reduce glutelin content, and remove globulin.

Usage and Biology

When OsGluB1 is mutated by CRISPR/Cas9 gene editing alone, the amino acid ratio of mutant glutelin B1 encoded by OsGluB1 is closer to the WHO recommended value than the wild type, that is, its biological value is higher, but it will not significantly affect the endosperm storage protein and lysine content of rice. When it is mutated with OsGlb at the same time, the expression of total protein, prolamine and lysine will increase, while the expression of glutelin and globulin will decrease.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

It is the target site of OsGluB1 gene in rice genome recognized by sgRNA of CRISPR/Cas9 gene editing system. It is located in the CDS region, upstream of PAM. After Cas9 protein cleaved the target gene guiding by sgRNA, the deletion of the second base g or the third base t of target gene leads to two different types of frameshift mutations in OsGluB1 gene, but these two kinds of mutations lead to the delayed appearance of stop codons. The mutant glutelin B1 encoded by OsGluB1 alone is hardly expressed. The molecular size of mutant glutelin B1 became larger, its hydrophobicity and instability increased, and the binding ability of human immunoglobulin E decreased.

Functional Parameters

protein	glutelin B1
target	cggttgccctgcgacttacc

This sgRNA target site sequence is located in the CDS. Its PAM is GGG. It is directly adjacent to PAM. Its CG content is 12 / 20.