Part:BBa_K3971003

gRNA target for Cpf1 based editing in the Neutral Site 1 of Synechococcus elongatus UTEX 2973.

Usage and Biology

This sequence is a gRNA target that is downstream of a TTC PAM sequence for Cpf1. This gRNA target sequence is to be cloned into the pSL2680 (Addgene Plasmid #85581) plasmid to make modifications in the Neutral site 1 of Synechococcus elongatus UTEX 2973, which it targets [1].

Neutral sites are loci in the genome with unknown functions and that can be disrupted without affecting cellular viability or causing any distinct phenotypes [2]. Cpf1 also known as Cas12a, can be used to make targeted modifications of DNA or RNA and is small in size, creating staggered instead of blunt cuts. It does not require tracrRNA and needs a 42-44 nucleotide crRNA. It also cuts distal to the PAM site without disrupting the target sequence and thus allows for future modifications at the same site [3].

References

1: Ungerer, Justin, and Himadri B. Pakrasi. "Cpf1 is a versatile tool for CRISPR genome editing across diverse species of cyanobacteria." Scientific reports 6.1 (2016): 1-9.

2: Pinto, Filipe, et al. "Improving a Synechocystis-based photoautotrophic chassis through systematic genome mapping and validation of neutral sites." DNA Research 22.6 (2015): 425-437.

3: Alok, Anshu, et al. "The rise of the CRISPR/Cpf1 system for efficient genome editing in plants." Frontiers in plant science 11 (2020): 264.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 13
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]