Part:BBa_J428062

Cas variants hSpCas9

hSpCas9

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 790
  Illegal PstI site found at 2212
  Illegal PstI site found at 2416
  Illegal PstI site found at 2446
  Illegal PstI site found at 3658
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 790
  Illegal PstI site found at 2212
  Illegal PstI site found at 2416
  Illegal PstI site found at 2446
  Illegal PstI site found at 3658
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 251
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 790
  Illegal PstI site found at 2212
  Illegal PstI site found at 2416
  Illegal PstI site found at 2446
  Illegal PstI site found at 3658
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 790
  Illegal PstI site found at 2212
  Illegal PstI site found at 2416
  Illegal PstI site found at 2446
  Illegal PstI site found at 3658
  Illegal NgoMIV site found at 1078
  Illegal NgoMIV site found at 2182
  Illegal NgoMIV site found at 2255
  Illegal NgoMIV site found at 2740
  Illegal NgoMIV site found at 3649
  
• 1000
  COMPATIBLE WITH RFC[1000]

iGEM Gifu 2023_Contribution

Group: iGEM Gifu 2023
Author: Miyu MAKIMURA

CRISPR MultiTargeter

We decided to use the CRISPR/Cas system, which is widely used as a knockout method, for our project. The general CRISPR/Cas system is a knockout system in which a single sgRNA is designed for a single target sequence selected from the target gene. To knock out variants of antibiotic resistance genes more efficiently, which exist in large numbers, we needed to design sgRNAs that could recognize and respond to multiple variants more efficiently. Therefore, we focused on CRISPR MultiTargeter (MultiTargeter), a sgRNA design tool [1]. MultiTargeter is a software program that allows you to design sgRNA sequences that recognize all input sequences by inputting multiple candidate target sequences. When multiple sequences are input into CRISPRMultiTargeter, algorithms are used to design sgRNAs that are unique or common to each of the multiple input sequences through sequence alignment, consensus sequence calculation, mismatch calculation, etc.

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Figure 1. sgRNA target search by CRISPR MultiTargeter

The sgRNAs designed by MultiTargeter has the potential to recognize and cleave a large number of target sequences with one or a small number of sgRNAs.An example of an actual application of MultiTargeter is its use to protect cotton against cotton leaf curl disease (CLCuD), a plant disease that infects cotton plants. MultiTargeter-designed sgRNAs have been used to identify SNP variants in in silico analysis of cannabinoid biosynthetic pathways introduced into cotton [2], [3].

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Reference

[1]. Prykhozhij SV, Rajan V, Gaston D, Berman JN. CRISPR multitargeter: a web tool to find common and unique CRISPR single guide RNA targets in a set of similar sequences. PLoS One. 2015 Mar 5;10(3):e0119372. doi: 10.1371/journal.pone.0119372. Erratum in: PLoS One. 2015;10(9):e0138634. PMID: 25742428; PMCID: PMC4351176.
[2]. Binyameen B, Khan Z, Khan SH, Ahmad A, Munawar N, Mubarik MS, Riaz H, Ali Z, Khan AA, Qusmani AT, Abd-Elsalam KA, Qari SH. Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus. Int J Mol Sci. 2021 Nov 21;22(22):12543. doi: 10.3390/ijms222212543. PMID: 34830426; PMCID: PMC8618328.
[3]. Matchett-Oates L, Braich S, Spangenberg GC, Rochfort S, Cogan NOI. In silico analysis enabling informed design for genome editing in medicinal cannabis; gene families and variant characterisation. PLoS One. 2021 Sep 22;16(9):e0257413. doi: 10.1371/journal.pone.0257413. PMID: 34551006; PMCID: PMC8457487.