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, 21 (10), 609-621

Modeling Disease In Vivo With CRISPR/Cas9


Modeling Disease In Vivo With CRISPR/Cas9

Lukas E Dow. Trends Mol Med.


The recent advent of CRISPR/Cas9-mediated genome editing has created a wave of excitement across the scientific research community, carrying the promise of simple and effective genomic manipulation of nearly any cell type. CRISPR has quickly become the preferred tool for genetic manipulation, and shows incredible promise as a platform for studying gene function in vivo. I discuss the current application of CRISPR technology to create new in vivo disease models, with a particular focus on how these tools, derived from an adaptive bacterial immune system, are helping us to better model the complexity of human cancer.

Keywords: CRISPR; Cas9; cancer; in vivo.


Figure 1
Figure 1. DNA repair mechanisms dictate genome editing outcomes
Following CRISPR/Cas9-induced DNA double-strand breaks, DNA is usually repaired by either homology directed repair (HDR) or non-homologous end joining (NHEJ). HDR enables integration of exogenous ‘repair templates’ such as single-stranded donor oligonucleotides (ssODN) or double-stranded DNA (dsDNA). NHEJ results in the generation of random insertions and deletions (indels) that may disrupt coding regions, or catalyze genomic rearrangements. The cellular preference for HDR or NHEJ following DNA damage can be augmented by small molecules that interfere with each mechanism, and thus bias toward the other. Abbreviations: ATM = Ataxia telangiectasia mutated; ATR = ataxia telangiectasia and Rad3-related; RAD51 = Rad51 recombinase; KU70 = XRCC6, X-ray repair complementing defective repair in Chinese hamster cells 6; KU80 = XRCC5, X-ray repair complementing defective repair in Chinese hamster cells 5; AZT = Azidothymidine; TFT = Trifluridine.
Figure 2
Figure 2. Genomic targeting of Cas9/sgRNA complexes
Genomic targeting of Cas9/sgRNA complexes is mediated by complementarity between the sgRNA (orange) and a genomic DNA (gDNA) target site (green) that lies immediately upstream of a protospacer adjacent motif (PAM, purple). For Cas9 derived from Streptococcus pyogenes, the PAM sequence is NGG, or less commonly, NAG. Cas9 variants from different bacterial species have alternative PAM requirements. Effective recognition and unwinding of the gDNA from the PAM results in double-strand cleavage by two independent nuclease domains in Cas9 (blue arrows).
Figure 3
Figure 3. CRISPR dominates papers published with genome editing technologies
Dotplot representing the average number of articles indexed on PubMed containing the search terms “CRISPR”, “TALEN”, or “ZFN” in the title or abstract, published over the last 10 years. Although it is the most recent addition to the genome editing toolbox, papers reporting CRISPR far outnumber both TALEN and ZFN based studies.

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