CRISPR System with AAV
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.
The sizelimitation 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 the 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 the DMD mouse model, thereby restoring the truncated but still functional Dystrophin.
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)