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Genome Editing

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  • Genome editing is enabled by the development of tools to make precise, targeted changes to the genome of living cells. Recently a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement. This follows several attempts over the years to manipulate gene function, including homologous recombination and RNA interference. RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function, but is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects. Other recent approaches to targeted genome modification -- zinc-finger nucleases [ZFNs] and transcription-activator like effector nucleases [TALENs]-- enable researchers to generate mutations by introducing double-stranded breaks to activate repair pathways. These approaches are costly and time consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.

    CRISPR (Clustered Regulary Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material.

    Cas9 in vivo: Bacterial adaptive immunity


    CRISPR loci in the bacterial genome are transcribed and processed into crRNA. Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence. (Figure not drawn to scale.)

    The simplicity of the CRISPR nuclease system, with only three components (Cas9 , crRNA and trRNA) makes this system amenable to adaptation for genome editing. By combining the crRNA and trRNA into a single synthetic guide RNA (sgRNA), a further simplified two component system can be used to introduce a targeted double stranded break. This break activates repair through error prone non-homologous end joining (NHEJ) or Homology directed Repair (HDR). In the presence of a donor template with homology to the targeted locus, the HDR pathway operates allowing for precise mutations to be made. In the absence of a template, NHEJ is activated resulting in insertions and/or deletions (indels) which disrupt the target locus.

    Genome engineering with Cas9 Nuclease


    Wild-type Cas9 nuclease site specifically cleaves double-stranded DNA activating double-strand break repair machinery. In the absence of a homologous repair template non-homologous end joining can result in indels disrupting the target sequence. Alternatively, precise mutations and knock-ins can be made by providing a homologous repair template and exploiting the homology directed repair pathway.

    Online Resources

    Plasmid Repositories:
    Addgene

    CRISPR-gRNA Design Tools:
    CRISPR Design
    ZiFiT
    CHOP CHOP

    Online Forums:
    Genome Engineering using CRISPR/Cas Systems

    Organism-specific Resources:
    Cas9-triggered homologous recombination
    Drosophila RNAi Screening Center at Harvard Medical School

    FAQs for Genome Editing

    Protocols for Genome Editing

      Publications related to Genome Editing:

    1. A-Bk Dad, S Ramakrishna, M Song, H Kim (2014). Enhanced gene disruption by programmable nucleases delivered by a minicircle vector. Gene Ther. , PubMedID: 25142139, DOI: 10.1038/gt.2014.76