Part:BBa_K3894015

nCas9
nCas9 is a mutation of Cas9 and is used to slice single-stranded DNA.

Description

nCas9 is a mutant of the Cas9 and used to generate ssDNA. In the CRISPR/Cas9 system, the nuclease domain of the Cas9 protein possesses DNA binding and cleavage functions, and is composed of two functional domains, HNH and RuvC. These two domains can individually cleave the complementary and non-complementary strands when directed by the guiding RNAs (gRNAs). Mutations in D10A and H840A of the Cas9 protein resulted in the inactivation of the RuvC and HNH domains, respectively, and abolished nuclease activity of Cas9 protein. The iGEM 2021 NEFU_China mutated Cas9 at the H840A site to wipe out the activity of the HNH domain and consequently generated the nCas9 with ssDNA cleavage activity^[1]. As a result, the nCas9-sgRNA complex specifically cleaves ssDNA at 3-bp upstream of the protospacer adjacent motif (PAM).

Experience

In this design, the iGEM 2021NEFU_China used nCas9 to make a single strand nick in the RPA products of the virus sequence, so that Phi29 can replace the ssDNA in the next step. In our studies, a 3-nucleotide mutations of the Cas9, a popular nuclease used to digest double-stranded DNA, becomes nCas9 that can only cleave ssDNA. A plasmid was constructed to in vitro transcribe sgRNAs that could guide nCas9 to find a specific site on a sequence derived from M13 geneIII. We successfully expressed and purified the nCas9 protein from E. coli (Figure 1).

Figure 1. SDS-PAGE analysis of purified recombinant nCas9 protein. The molecular weight of the nCas9 protein is 162.3 kDa. | 1 and 2. Eluent of His×6-tagged nCas from Ni-NTA agarose beads.

Figure 2. Verification of nCas9 nickase activity of target plasmids. |1-2. Linear fragments, standard plasmids and nicked plasmids.

The iGEM 2021 NEFU_China verified the cleavage activity of nCas9 by detecting the position of plasmid bands in the gel (Figure 2). The nickase activity of the nCas9 was determined by its ability of creating nicked DNA within a reaction time of 30 min. The target plasmid carrying the geneIII fragment and streptomycin resistant gene was transformed bacteria, and the constructed plasmid was extracted and analyzed by agarose gel electrophoresis.The nicked plasmid after cleaved by nCas9 showed a slowly migrated band compared to that of the intact plasmid in the agarose gel. Based on the analysis of the agarose gel, we can preliminarily conclude that the figure shows that nCas9 possesses the activity of generating nicked DNA.

References

[1] Liu Y , Tao W , Wen S , et al. In Vitro CRISPR/Cas9 System for Efficient Targeted DNA Editing[J]. mBio, 2015, 6(6):e01714-15.

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]