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Gene Editing

Use CRISPR-Cas9 systems with Custom RNA Guides for Gene Editing Applications

Many bacterial and archaeal CRISPR-Cas systems have been identified with diverse sets of mechanisms, Cas proteins, and multi-subunit complexes. In particular, the processes and key components of the Streptococcus pyogenes CRISPRCas9 system have been well-studied and adapted for genome engineering in mammalian cells. In S. pyogenes, only three components are required for targeted DNA cleavage at specific target sites adjacent to a PAM: (1) The endonuclease Cas9, programmed by (2) a mature crRNA processed from transcription of the CRISPR locus/array which complexes with (3) another CRISPR locus-encoded RNA, the trans-activating RNA (tracrRNA).

Upon site-specific double-stranded DNA cleavage, a mammalian cell can repair such a break through either nonhomologous end joining (NHEJ) or homologous recombination (HR). NHEJ is often imperfect, resulting in small insertions and deletions (indels) that can result in nonsense mutations and truncation of protein products or introduction of a stop codon to produce gene knockouts. This endogenous DNA break repair process, coupled with the highly tractable S. pyogenes CRISPR-Cas9 system, allows for a readily engineered platform to permanently disrupt gene function.

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