1. Fig. 1 Timeline of CRISPR-Cas and genome engineering research fields
Fig. 1 Timeline of CRISPR-Cas and genome engineering research fields.Key developments in both fields are shown.
Timeline of CRISPR-Cas and genome engineering research fields.Key developments in both fields are shown. These two fields merged in 2012 with the discovery that Cas9 is an RNA-programmable DNA endonuclease, leading to the explosion of papers beginning in 2013 in which Cas9 has been used to modify genes in human cells as well as many other cell types and organisms.
Jennifer A. Doudna, and Emmanuelle Charpentier Science 2014;346:
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2. Fig. 2 Biology of the type II-A CRISPR-Cas system
Fig. 2 Biology of the type II-A CRISPR-Cas system.The type II-A system from S. pyogenes is shown as an example.
Biology of the type II-A CRISPR-Cas system.The type II-A system from S. pyogenes is shown as an example. (A) The cas gene operon with tracrRNA and the CRISPR array. (B) The natural pathway of antiviral defense involves association of Cas9 with the antirepeat-repeat RNA (tracrRNA:crRNA) duplexes, RNA co-processing by ribonuclease III, further trimming, R-loop formation, and target DNA cleavage. (C) Details of the natural DNA cleavage with the duplex tracrRNA:crRNA.
Jennifer A. Doudna, and Emmanuelle Charpentier Science 2014;346:
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3. Fig. 3 Evolution and structure of Cas9. The structure of S
Fig. 3 Evolution and structure of Cas9.The structure of S. pyogenes Cas9 in the unliganded and RNA-DNA–bound forms [from (77, 81)].
Evolution and structure of Cas9.The structure of S. pyogenes Cas9 in the unliganded and RNA-DNA–bound forms [from (77, 81)].
Jennifer A. Doudna, and Emmanuelle Charpentier Science 2014;346:
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4. Fig. 4 CRISPR-Cas9 as a genome engineering tool
Fig. 4 CRISPR-Cas9 as a genome engineering tool.(A) Different strategies for introducing blunt double-stranded DNA breaks into genomic loci, which become substrates for endogenous cellular DNA repair machinery that catalyze nonhomologous end joining (NHEJ) or homology-directed repair (HDR).
CRISPR-Cas9 as a genome engineering tool.(A) Different strategies for introducing blunt double-stranded DNA breaks into genomic loci, which become substrates for endogenous cellular DNA repair machinery that catalyze nonhomologous end joining (NHEJ) or homology-directed repair (HDR). (B) Cas9 can function as a nickase (nCas9) when engineered to contain an inactivating mutation in either the HNH domain or RuvC domain active sites. When nCas9 is used with two sgRNAs that recognize offset target sites in DNA, a staggered double-strand break is created. (C) Cas9 functions as an RNA-guided DNA binding protein when engineered to contain inactivating mutations in both of its active sites. This catalytically inactive or dead Cas9 (dCas9) can mediate transcriptional down-regulation or activation, particularly when fused to activator or repressor domains. In addition, dCas9 can be fused to fluorescent domains, such as green fluorescent protein (GFP), for live-cell imaging of chromosomal loci. Other dCas9 fusions, such as those including chromatin or DNA modification domains, may enable targeted epigenetic changes to genomic DNA.
Jennifer A. Doudna, and Emmanuelle Charpentier Science 2014;346:
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5. Fig. 5 Examples of cell types and organisms that have been engineered using Cas9.
Jennifer A. Doudna, and Emmanuelle Charpentier Science 2014;346:
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6. Fig. 6 Future applications in biomedicine and biotechnology
Fig. 6 Future applications in biomedicine and biotechnology.Potential developments include establishment of screens for target identification, human gene therapy by gene repair and gene disruption, gene disruption of viral sequences, and programmable RNA targeting.
Future applications in biomedicine and biotechnology.Potential developments include establishment of screens for target identification, human gene therapy by gene repair and gene disruption, gene disruption of viral sequences, and programmable RNA targeting.
Jennifer A. Doudna, and Emmanuelle Charpentier Science 2014;346:
Published by AAAS

7. David Baltimore et al. Science 2015;348:36-38
Published by AAAS

8. How endonuclease gene drives spread altered genes through populations
How endonuclease gene drives spread altered genes through populations.(A) Altered genes (blue) normally have a 50% chance of being inherited by offspring when crossed with a wild-type organism (gray).
How endonuclease gene drives spread altered genes through populations.(A) Altered genes (blue) normally have a 50% chance of being inherited by offspring when crossed with a wild-type organism (gray). (B) Gene drives can increase this chance to nearly 100% by cutting homologous chromosomes lacking the alteration, which can cause the cell to copy the altered gene and the drive when it fixes the damage. (C) By ensuring that the gene is almost always inherited, the gene drive can spread the altered gene through a population over many generations, even if the associated trait reduces the reproductive fitness of each organism. The recently developed CRISPR nuclease Cas9, now widely used for genome engineering, may enable scientists to drive genomic changes that can be generated with Cas9 through sexually reproducing organisms (1).
Kenneth A. Oye et al. Science 2014;345:
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9. Kenneth A. Oye et al. Science 2014;345:626-628
Published by AAAS