“신제품 EnGen® Seq1 Cas9은 이중 가닥 DNA(dsDNA)의 특정 부위 절단을 촉매 하는 RNA-guided DNA endonuclease입니다.“
- EnGen® Mutation Detection Kit (NEB #E3321S)와 호환 가능
- Product Source: An E. coli strain that carries the cloned Cas9 gene from Streptococcus equinus with N- and C-terminal Simian virus 40 (SV40) T antigen nuclear localization signal (NLS) and a C-terminal 6XHis tag.
- 5´- NAGA -3´ PAM sequence allows targeting of additional genomic regions
- Ideal for direct introduction of Cas9/sgRNA complexes
- Active in in vitro reactions from 20°C to 45°C
|
Component Name |
Temp |
Amount |
Concentration |
|
EnGen® Seq1 Cas9 |
-20 ℃ |
1 x 500 pmol |
20 µM |
| NEBuffer™ r3.1 |
-20 ℃ |
1 x 1.25 ml |
10X |
Figure 1. Schematic representation of S. equinus Cas9 nuclease complexed with a single guide RNA and target DNA
S. equinus Cas9 (Seq1 Cas9) protein is shown in beige, single guide RNA (sgRNA) is depicted in blue, and the DNA is shown in grey, green, and red. The DNA target, or protospacer, is shown in green and the base pairing complementarity of the target strand with the guide RNA in the R-loop is illustrated. The protospacer adjacent motif (PAM) which is required for Cas9 binding to DNA is shown in red and the locations of DNA strand cleavage are indicated with black triangles.
Cas9 nuclease 비교
|
Cat# |
Description |
PAM sequence1) |
Source |
|
NEB #M0668 (신제품) |
EnGen Seq1 Cas9 |
5’-NAGA-3’ |
E. coli strain that carries the cloned Cas9 gene from Streptococcus equinus |
|
EnGen Sau Cas9 |
5’-NNGRRT-3’ |
E. coli strain that carries the cloned Cas9 gene from Staphylococcus aureus |
|
|
EnGen Spy Cas9 NLS |
5’-NGG-3’ |
E. coli strain that carries the cloned Cas9 gene from Streptococcus pyogenes |
1) Cas9이 PAM 염기서열을 인지하면, Cas9에 의해 DNA의 이중쇄절단(Double strand break; DSB)이 발생
Q&A
EnGen Seq1 Cas9 contains two nuclear localization signals located on the N- and C-termini of the protein.
Yes, both of these products can be used to detect genome editing events generated with EnGen Seq1 Cas9.
The single guide RNA for use with EnGen Seq1 Cas9 has the following sequence:
5’- NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUGUGUUGGAAACAACACAGCGAGUUAAAAUAAGGCUUUGUCCGUACACAACUUGUAAAAGUGGCACCCGAUUCGGGUGCAUUUUUU -3’
The underlined 20-nucleotide spacer sequence is specific to the desired DNA target region immediately upstream of the 5’-NAGA-3’ protospacer adjacent motif. The spacer sequence does not contain the PAM sequence itself. The portion of the sgRNA not underlined is the scaffold specific for ribonucleoprotein complex formation with EnGen Seq1 Cas9.
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