Out of the many challenges scientists face in bringing genomic medicine to patients, delivery is near the top of the list. Due to these barriers in getting the genetic payload to the correct target cells, current methods have been restricted to ex vivo approaches or in vivo liver editing. In addition, first-generation CRISPR systems (Cas9 and Cas12a) are too large for efficient in vivo delivery via a single adeno-associated viral (AAV) vector..

在科学家为患者带来基因组医学所面临的众多挑战中，分娩几乎排在首位。由于在将遗传有效载荷获得正确的靶细胞方面存在这些障碍，目前的方法仅限于离体方法或体内肝脏编辑。此外，第一代CRISPR系统（Cas9和Cas12a）太大，无法通过单个腺相关病毒（AAV）载体进行有效的体内递送。。

Now, new preclinical research presents the discovery and engineering of NanoCas—an ultracompact CRISPR nuclease capable of extending CRISPR’s reach in vivo beyond liver targets. NanoCas is the first ultracompact CRISPR system capable of efficient extrahepatic editing when delivered systemically using a single AAV vector..

现在，新的临床前研究提出了NanoCas的发现和工程化，NanoCas是一种超紧凑的CRISPR核酸酶，能够将CRISPR在体内的范围扩展到肝脏靶标之外。NanoCas是第一个超紧凑型CRISPR系统，当使用单个AAV载体全身递送时，能够进行有效的肝外编辑。。

This work is published on

这项工作发表于

bioRxiv

生物Rxiv

in a preprint titled, “

在标题为“的预印本中

Single-AAV CRISPR editing of skeletal muscle in non-human primates with NanoCas, an ultracompact nuclease

使用超紧凑核酸酶NanoCas对非人灵长类动物骨骼肌进行单一AAV CRISPR编辑

.”

.”

NanoCas is a novel Cas enzyme (approximately one-third the size of Cas9) that can be accommodated within a single AAV vector while leaving room for additional payloads such as regulatory elements, guide RNAs, or non-double strand break editing machinery. This could be utilized for techniques such as reverse transcriptase editing, base editing, and epigenetic editing..

NanoCas是一种新型的Cas酶（大约是Cas9大小的三分之一），可以容纳在单个AAV载体中，同时为额外的有效载荷（例如调控元件，指导RNA或非双链断裂编辑机制）留下空间。这可以用于逆转录酶编辑，碱基编辑和表观遗传编辑等技术。。

The research group at Mammoth Biosciences—a Brisbane, CA-based company—screened 176 ultracompact CRISPR systems found in metagenomic data and applied protein engineering approaches to enhance the editing efficiency of NanoCas. The optimized NanoCas, the authors say, “exhibits potent editing capabilities across various cell systems and tissues in vivo when administered via adeno-associated viral (AAV) vectors.”.

位于加利福尼亚州布里斯班的Mammoth Biosciences公司的研究小组筛选了在宏基因组数据中发现的176个超紧凑CRISPR系统，并应用了蛋白质工程方法来提高NanoCas的编辑效率。作者说，优化后的NanoCas“当通过腺相关病毒（AAV）载体给药时，在体内各种细胞系统和组织中表现出强大的编辑能力。”。

When targeting the PCSK9 gene in mouse liver in vivo, NanoCas showed saturating editing efficiencies of approximately 60%, on par with that of SaCas9, which is about three-fold larger in size. Both CRISPR systems reduced serum PCSK9 protein to undetectable levels.

当在体内靶向小鼠肝脏中的PCSK9基因时，NanoCas显示出约60%的饱和编辑效率，与SaCas9相当，SaCas9的大小约为SaCas9的三倍。两种CRISPR系统均将血清PCSK9蛋白降低至检测不到的水平。

NanoCas also demonstrated 10% to 40% editing of the dystrophin gene across the quadricep, calf, and heart muscle in a humanized mouse model of Duchenne Muscular Dystrophy (DMD), when delivered via a single AAV vector.

当通过单个AAV载体递送时，NanoCas还证明了在杜兴氏肌营养不良症（DMD）的人源化小鼠模型中，肌营养不良蛋白基因在股四头肌，小腿和心肌中的编辑率为10%至40%。

Lastly, NanoCas achieved in vivo editing efficiencies of up to 30% when targeting dystrophin in the skeletal muscle of cynomolgus macaques. NanoCas also showed 15% editing across the heart, compared to 10% with SaCas9. And analysis of liver tissue showed minimal off-target editing.

。NanoCas在整个心脏中也显示出15%的编辑，而SaCas9的编辑率为10%。肝组织分析显示最小的脱靶编辑。

“Potent editing of extrahepatic tissues in vivo has been a roadblock for the gene editing field,” said Trevor Martin, PhD, co-founder and CEO of Mammoth Biosciences. “NanoCas’ compact size makes it compatible with a wide range of gene editing modalities—including base editing, reverse transcriptase editing, and epigenetic modification—while still allowing for delivery using a single AAV vector.

Mammoth Biosciences联合创始人兼首席执行官TrevorMartin博士说：“体内肝外组织的有效编辑一直是基因编辑领域的障碍。”。“NanoCas的紧凑尺寸使其与多种基因编辑方式兼容，包括碱基编辑，逆转录酶编辑和表观遗传修饰，同时仍允许使用单个AAV载体进行递送。

This study is a major step toward enabling any edit to be made in any cell in vivo, thereby dramatically increasing the number of patients who could benefit from genetic medicines and delivering on the full promise of CRISPR.”.

这项研究是朝着在体内任何细胞中进行任何编辑迈出的重要一步，从而大大增加了可以从遗传药物中受益的患者数量，并实现了CRISPR的全部承诺。”。

News

新闻

Adeno associated viral vector

腺相关病毒载体

CRISPR-Cas9 technology

CRISPR-Cas9技术

Nanotechnology

纳米技术

Primates

灵长类

Skeletal muscle

骨骼肌