Coding

Part:BBa_K4687000

Designed by: Yiming Jiang   Group: iGEM23_HBUT-China   (2023-09-30)


CRISPR-MAD7 nuclease

We constructed the CRISPR-MAD7 gene editing system by altering the strong promoter in the sequence of the gene encoding the CRISPR-MAD7 nuclease. We constructed a single-plasmid system for CRISPR-MAD7 nuclease editing by inserting CRISPR-MAD7 nuclease sequences into different vector skeletons to edit genes in Corynebacterium glutamicum. In addition, we guided CRISPR-MAD7 nuclease to recognize different PAM sites by altering gRNA to improve the editing efficiency of the CRISPR-MAD7 gene editing system in valley rod. In our project, the system was mainly used for targeted gene knockouts, and we used CRISPR-MAD7 nuclease to build our biobricks.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 650
    Illegal BglII site found at 698
    Illegal BglII site found at 1019
    Illegal BglII site found at 2234
    Illegal BglII site found at 2886
    Illegal BglII site found at 3740
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1801
  • 1000
    COMPATIBLE WITH RFC[1000]



Introduction

CRISPR-MAD7 is a nuclease that belongs to the Class 2 type V-A CRISPR family (Cas12a-like). It was identified in Eubacterium rectale. It was publicly released as a nuclease freely available for both academic and commercial use. The system recognizes sites with T-rich PAM sequences (YTTN). Similar to CRISPR-Cas12a (Tang et al.,2017), CRISPR-MAD7 is effective in creating indels (insertion/deletion), mainly of 6-10 bp and so may be suitable for inactivating both genes and regulatory elements. [1]

CRISPR-MAD7 is a CRISPR nuclease based on a codon-optimized gene from the Eubacterium rectale (refseq WP_055225123.1). The codon-optimized CRISPR-MAD7 gene shows 76% identity to the native E. rectale nucleotide sequence and encodes for a monomeric protein composed by 1263 amino acid residues with a molecular weight of 147.9kDa. [2]

Overall tertiary structure of different perspectives of CRISPR-MAD7.
Figure 1. Overall tertiary structure of different perspectives of CRISPR-MAD7.

Usage and Biology

We constructed the CRISPR-MAD7 gene editing system by altering the strong promoter in the sequence of the gene encoding the CRISPR-MAD7 nuclease. We constructed a single-plasmid system for CRISPR-MAD7 nuclease editing by inserting CRISPR-MAD7 nuclease sequences into different vector skeletons to edit genes in Corynebacterium glutamicum. In addition, we guided CRISPR-MAD7 nuclease to recognize different PAM sites by altering gRNA to improve the editing efficiency of the CRISPR-MAD7 gene editing system in valley rod. In our project, the system was mainly used for targeted gene knockouts, and we used CRISPR-MAD7 nuclease to build our biobricks.

Characterization

Stable expression among clones

From left to right, the transformed plasmids were pJYS1Peftu,pJYS1-MAD7-ΔcrtI,pJYS1-MAD7-ΔcrtE:crtR As can be seen in the figure, the original vector backbone gene without editing by CRISPR-MAD7 system was expressed in C. glutamicum, making the colony color all yellow. The expression of the CRISPR-MAD7 gene knockout gene in C. glutamicum showed a yellow-white colony color. The expression of the double gene knockout by CRISPR-MAD7 gene editing system in C. glutamicum showed red colony color.
Figure 2. From left to right, the transformed plasmids were pJYS1Peftu, pJYS1-MAD7-ΔcrtI, pJYS1-MAD7-ΔcrtE:crtR As can be seen in the figure, the original vector backbone gene without editing by CRISPR-MAD7 system was expressed in C. glutamicum, making the colony color all yellow. The expression of the CRISPR-MAD7 gene knockout gene in C. glutamicum showed a yellow-white colony color. The expression of the double gene knockout by CRISPR-MAD7 gene editing system in C. glutamicum showed red colony color.

Reference

  1. ↑ Qiupeng Lin,Zixu Zhu,Guanwen Liu,Chao Sun,Dexing Lin,Chenxiao Xue… & Jin-Long Qiu.(2021).Genome editing in plants with MAD7 nuclease. Journal of Genetics and Genomics(06),444-451.
  2. ↑ Garcia K V,Haar K J R,Dorota Z J, et al. A Mad7 System for Genetic Engineering of Filamentous Fungi[J]. Journal of Fungi,2022,9(1).
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