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CRISPR System with AAV

CRISPR, an immune system discovered in bacteria, has been successfully adapted into an eukaryote genome-editing tool in 2013 and has been extensively applied to basic and clinic research in recent years ^{(1, 2, 3)}. Since the SpCas9 knock-in mouse has been created and AAV-friendly SaCas9 and NmCas9 have been further characterized ^{(4, 5)}, the combination of AAV and Cas9 has become a more versatile tool for animal experimentation ^{(6, 7)}.

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SpCas9

Gene-Knockout

Zhang’s group from the Broad Institute (MIT) breed a SpCas9 knock-in mouse that can express SpCas9 constitutively or Cre-dependently in cells of whole body, and demonstrated that utilizing AAV delivering sgRNA and donor sequence can precisely modify the KRAS gene into a tumor-causing mutation, which then results in rapid tumor growth in 1 to 3 months ⁽⁵⁾. SpCas9 mouse is now available from Jackson Lab.

Figures from Ref. 5

Gene-Activation

Konermann S.’s group (2015) from MIT published their discovery that 15-bp short sgRNA can arrest a wild-type SpCas9 at the target locus without cutting the DNA ⁽¹¹⁾. Thus, when the sgRNA scaffold bearing two additional loops (sgRNA-MS2) binds a fusion of activation components MS2-P65-HSF1, the gene will be activated if the sgRNA targets the transcription start region. This finding shows a novel way to overcome AAV size-limitation for large gene over-expression. Zhang et al. found a similar modification that also worked for SaCas9^(12).

Figures from Ref. 11

We have developed a set of sgRNA scaffold-optimized entry plasmids for creating the fit constructs for your own purpose. (click here)

SaCas9

The size-limitation of AAV is about 5 Kbp, so the length of SpCas9 (4.3 Kbp) is relatively big and not suitable for flexible manipulation such as switching appropriate promoters for different target organs. For the broader application with AAV, Zhang’s group characterized and applied SaCas9 (3.3 Kbp), which can fit into the AAV with 1 Kbp more space for specific expression regulation compared to SpCas9. This was used to specifically express SaCas9 in liver to knockout a Pcsk9 gene, and 40% target gene modification was observed in this case.

In most cases, using the AAV carrying SaCas9 will eliminate the need for SpCas9-mouse lines, thus potentially saving a lot of time on crossbreeding proper mouse lines for studies using disease mouse models.

On the final day of 2015, three groups published their promising results on utilizing AAV to deliver Cas9 to therapy in DMD mouse as a therapeutics published in Science^{(8, 9, and 10)}. All three groups applied the same strategy: injection of AAV expressing SaCas9/SpCas9 and sgRNAs to delete the mutated Exon 23 in DMD mouse model, thereby restoring the truncated but still functional Dystrophin.

Figures from Ref. 9

For researchers willing to apply this mighty technology in research, PackGene provides a set of optimized entry plasmids.

REFFERENCES

1. Martin Jinek et al. Science 337, 816 (2012); DOI: 10.1126/science.1225829
2. Le Cong et al. Science 339,819 (2013); DOI: 10.1126/science.1231143
3. Prashant Mali et al. Science 339,823 (2013); DOI: 10.1126/science.1232033
4. Randall J. Platt et al. Cell 159, 1–16 (2014); DOI: 10.1016/j.cell.2014.09.014
5. F. Ann Ran et al. Nature 520, 186 (2015); doi:10.1038/nature14299
6. Florian Schmidt and Dirk Grimm. Biotechnology J. 10, 258 (2015); DOI0.1002/biot.201400529
7. David B. T. Cox et al. Nature Medicine 21,121 (2015); doi: 10.1038/nm.3793.
8. C. Long et al., Science 10.1126/science.aad5725 (2015)
9. C. E. Nelson et al., Science 10.1126/science.aad5143 (2015)
10. M. Tabebordbar et al., Science 10.1126/science.aad5177 (2015).
11. Dahlman JE et al., Nat Biotechnol. 2015 Nov;33(11):1159-61.
12. Nishimasu et al., Cell 162, 1113–1126 (2015)