Tran Mai H, Park Hajeung, Nobles Christopher L, Karunadharma Pabalu, Pan Li, Zhong Guocai, Wang Haimin, He Wenhui, Ou Tianling, Crynen Gogce, Sheptack Kelly, Stiskin Ian, Mou Huihui, Farzan Michael
Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA.
X-ray Crystallography Core, The Scripps Research Institute, Jupiter, FL 33458, USA.
Mol Ther Nucleic Acids. 2021 Feb 18;24:40-53. doi: 10.1016/j.omtn.2021.02.012. eCollection 2021 Jun 4.
CRISPR effector proteins introduce double-stranded breaks into the mammalian genome, facilitating gene editing by non-homologous end-joining or homology-directed repair. Unlike the more commonly studied Cas9, the CRISPR effector protein Cas12a/Cpf1 recognizes a T-rich protospacer adjacent motif (PAM) and can process its own CRISPR RNA (crRNA) array, simplifying the use of multiple guide RNAs. We observed that the Cas12a ortholog of MA2020 (Lb2Cas12a) edited mammalian genes with efficiencies comparable to those of AsCas12a and LbCas12a. Compared to these well-characterized Cas12a orthologs, Lb2Cas12a is smaller and recognizes a narrow set of PAM TTTV. We introduced two mutations into Lb2Cas12a, Q571K and C1003Y, that increased its cleavage efficiency for a range of target sequences beyond those of the commonly used Cas12a orthologs AsCas12a and LbCas12a. In addition to the canonical TTTV PAM, this variant, Lb2-KY, also efficiently cleaved target regions with CTTN PAMs. Finally, we demonstrated that Lb2-KY ribonucleoprotein (RNP) complexes edited two hemoglobin target regions useful for correcting common forms of sickle-cell anemia more efficiently than commercial AsCas12a RNP complexes. Thus, Lb2-KY has distinctive properties useful for modifying a range of clinically relevant targets in the human genome.
CRISPR效应蛋白会在哺乳动物基因组中引入双链断裂，通过非同源末端连接或同源定向修复促进基因编辑。与更常被研究的Cas9不同，CRISPR效应蛋白Cas12a/Cpf1识别富含T的原间隔相邻基序（PAM），并能加工自身的CRISPR RNA（crRNA）阵列，从而简化了多个引导RNA的使用。我们观察到MA2020的Cas12a直系同源物（Lb2Cas12a）编辑哺乳动物基因的效率与AsCas12a和LbCas12a相当。与这些特征明确的Cas12a直系同源物相比，Lb2Cas12a更小，并且识别一组狭窄的PAM TTTV。我们在Lb2Cas12a中引入了两个突变，即Q571K和C1003Y，这提高了它对一系列靶序列的切割效率——超出了常用的Cas12a直系同源物AsCas12a和LbCas12a的切割效率范围。除了典型的TTTV PAM外，这个变体Lb2-KY还能有效切割具有CTTN PAM的靶区域。最后，我们证明，Lb2-KY核糖核蛋白（RNP）复合物编辑两个对纠正常见形式的镰状细胞贫血有用的血红蛋白靶区域，比商业化的AsCas12a RNP复合物更有效。因此，Lb2-KY具有独特的特性，有助于修饰人类基因组中一系列临床相关的靶点。
