Hirano Hisato, Gootenberg Jonathan S, Horii Takuro, Abudayyeh Omar O, Kimura Mika, Hsu Patrick D, Nakane Takanori, Ishitani Ryuichiro, Hatada Izuho, Zhang Feng, Nishimasu Hiroshi, Nureki Osamu
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
Cell. 2016 Feb 25;164(5):950-61. doi: 10.1016/j.cell.2016.01.039. Epub 2016 Feb 11.
The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5'-NGG-3' PAM, and used the structural information to create a variant that can recognize the more relaxed 5'-YG-3' PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5'-YG-3' PAM, thus expanding the target space of the CRISPR-Cas9 toolbox.
RNA引导的核酸内切酶Cas9可切割与引导RNA互补的双链DNA靶标，并已应用于可编程基因组编辑。Cas9介导的切割需要一个与DNA靶标序列并列的原间隔相邻基序（PAM），从而限制了可靶向位点的范围。在此，我们报告了来自新凶手弗朗西斯菌（FnCas9）的Cas9的1.7 Å分辨率晶体结构，FnCas9是最大的Cas9直系同源物之一，它与引导RNA及其含PAM的DNA靶标形成复合物。FnCas9与其他Cas9直系同源物的结构比较揭示了远缘相关的CRISPR-Cas9系统之间显著的保守和差异特征。我们发现FnCas9识别5'-NGG-3' PAM，并利用结构信息创建了一种可识别更宽松的5'-YG-3' PAM的变体。此外，我们证明FnCas9核糖核蛋白复合物可显微注射到小鼠受精卵中，以编辑具有5'-YG-3' PAM的内源性位点，从而扩展了CRISPR-Cas9工具箱的靶标空间。
