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Genome Editing

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 Regularly 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.

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Cas9 in vivo: Bacterial adaptive immunity

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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

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FAQs for Genome Editing
Protocols for Genome Editing
Application Notes for Genome Editing
    Publications related to Genome Editing
    • Zheng Hu, Wencheng Ding, Da Zhu, Lan Yu, Xiaohui Jiang, Xiaoli Wang, Changlin Zhang, Liming Wang, Teng Ji, Dan Liu, Dan He, Xi Xia, Tao Zhu, Juncheng Wei, Peng Wu, Changyu Wang, Ling Xi, Qinglei Gao, Gang Chen, Rong Liu, Kezhen Li, Shuang Li, Shixuan Wang, Jianfeng Zhou, Ding Ma, Hui Wang (2015) TALEN-mediated targeting of HPV oncogenes ameliorates HPV-related cervical malignancy. J Clin Invest; 125, 425-36. PubMedID: 25500889, DOI: 10.1172/JCI78206
    • 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
Online Resources

Online Resources

Plasmid Repositories:
Addgene

CRISPR-gRNA Design Tools:
Desktop Genetics
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

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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.


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