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Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications.
In the I-E system of E. coli, Cas1 and Cas2 form a complex where a Cas2 dimer bridges two Cas1 dimers. [116] In this complex, Cas2 performs a non-enzymatic scaffolding role, [ 116 ] binding double-stranded fragments of invading DNA, while Cas1 binds the single-stranded flanks of the DNA and catalyses their integration into CRISPR arrays.
But Cas9 will not cleave the protospacer sequence unless there is an adjacent PAM sequence. The spacer in the bacterial CRISPR loci will not contain a PAM sequence, and thus will not be cut by the nuclease, but the protospacer in the invading virus or plasmid will contain the PAM sequence, and thus will be cleaved by the Cas9 nuclease. [4]
CRISPR-associated transposons are similar to the Tn7 transposon which functions with a cut and paste mechanism. [1] It contains a heteromeric transposase consisting of TnsA and TnsB proteins, and a regulator protein TnsC. [1]
Cas9 Endonuclease Dead, also known as dead Cas9 or dCas9, is a mutant form of Cas9 whose endonuclease activity is removed through point mutations in its endonuclease domains. Similar to its unmutated form, dCas9 is used in CRISPR systems along with gRNAs to target specific genes or nucleotides complementary to the gRNA with PAM sequences that ...
CRISPR-Cas9 genome editing techniques have many potential applications. The use of the CRISPR-Cas9-gRNA complex for genome editing [10] was the AAAS's choice for Breakthrough of the Year in 2015. [11] Many bioethical concerns have been raised about the prospect of using CRISPR for germline editing, especially in human embryos. [12]
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Cas1 is a metal-dependent DNA-specific endonuclease that produces double-stranded DNA fragments. [1] Cas1 forms a stable complex with the other universally conserved CRISPR-associated protein, cas2 , which is essential to spacer acquisition for CRISPR systems.