复制子: p15A ori
pCas9可在大肠杆菌中表达Cas9蛋白，和pCRISPR搭配可用于基因敲除，低拷贝质粒，Bacterial expression of Cas9 nuclease, tracrRNA and crRNA guide.
pCas9 can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-baseddirected evolution, and combinatorial design. CRISPR-Cas9 nucleases enable efficient genome editing in a wide variety of organisms and cell types . Target site recognition by Cas9 is programmed by a chimeric single guide RNA (sgRNA) that encodes a sequence complementary to a target protospacer5 , but also requires recognition of a short neighboring PAM . SpCas9, the most robust and widely used Cas9 to date, primarily recognizes NGG PAMs and is consequently restricted to sites that contain this motif . It can therefore be challenging to implement genome editing applications that require precision, such as: homology-directed repair (HDR), which is most efficient when DSBs are placed within 10-20 bps of a desired alteration; the introduction of variable-length insertion or deletion (indel) mutations into small size genetic elements such as microRNAs, splice sites, short open reading frames, or transcription factor binding sites by non-homologous end-joining (NHEJ); and allele-specific editing, where PAM recognition might be exploited to differentiate alleles.