Part:BBa_K4343130

TEFin-nSpRYCBE

 A cytosine base editor with GAP promoter-driven expression in which the Cas9 protein is nSpRY. This system creates a gap at the targeting site and facilitates the efficiency of base editing in Y.lipolytica. More critically, SpRY is a PAM-less Cas protein that can be targeted and edited at any location within a gene. At the same time, it is ensured that the editing efficiency is high enough to obtain mutants.

Background and Introduction

 In contrast to the homologous directed repair (HDR) pathway, the oleaginous yeast Yarrowia lipolytica prefers the non-homologous end-joining (NHEJ) pathway as a double-strand break (DSB) repair system. Due to this selectivity for the DNA repair mechanism, it is difficult to use homologous recombination to remove important genes or insert foreign genes into certain places precisely. In recent years, a base editing system based on the CRISPR/Cas9 system has been developed, which can create nonsense mutations within the target gene to precisely block gene expression without the need for DSB repair. It works by a nucleotide deaminase being fused to dead Cas9 (dCas9), which is recruited to a specific DNA site via gRNA. Among the many base editor tools, the Target-AID system, based on Petromyzon marinus Cytidine deaminase (PmCDA1), has been efficiently used in a variety of organisms such as Saccharomyces cerevisiae, Escherichia coli and mammalian cells (Edit window range of -15 to -20bp) [2-4]. This component (BioBrick BBa_K3645011) was first registered by iGEM20_Peking Team in 2020 (Fig 1).
 We are trying to improve the ability of BioBrick BBa_K3645011 to work more efficiently in Y.lipolytica by using optimised related components. We first supposed whether replacing dCas9 with nCas9 will increase the targeting and editing efficiency of CBE. We also hypothesized that the expression level of CBE could be increased through using the stronger promoters thus increasing editing efficiency. For this,the improving of BioBrick BBa_K3645011 are divided into three sections as follows: (i) the replacement of dCas9 by nick Cas9 (nCas9), (ii) promoter optimization and (iii) Cas9 without protospacer adjacent motif (PAM) limitation (SpRY)[5] to expand the editing range. 

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1538
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4865
  Illegal BamHI site found at 3817
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 426
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1
  Illegal BsaI site found at 429
  Illegal BsaI site found at 5443
  Illegal BsaI.rc site found at 423
  Illegal BsaI.rc site found at 4552
  Illegal BsaI.rc site found at 5707