Orthogonal CRISPR-Cas tools for genome editing, inhibition, and CRISPR recording in zebrafish embryos.
Orthogonal CRISPR-Cas tools for genome editing, inhibition, and CRISPR recording in zebrafish embryos.
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DOI：
10.1093/genetics/iyab196
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发表时间：
2021-11
期刊：
影响因子：
3.3
通讯作者：
Paige Takasugi;Shengzhou Wang;Kimberly T Truong;Evan P. Drage;Sahar N. Kanishka;Marissa A. Higbee
中科院分区：
文献类型：
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作者：
Paige Takasugi;Shengzhou Wang;Kimberly T Truong;Evan P. Drage;Sahar N. Kanishka;Marissa A. Higbee
CRISPR-Cas的世界还在继续扩大。来自化脓性链球菌（Streptococcus pyogenes）的II型CRISPR-Cas系统（SpyCas9）因其在细胞和生物体中的高效率而被广泛用于基因组编辑。然而，专注于单一的CRISPR-Cas系统对目标选择和多路基因组工程施加了限制。我们假设源自不同细菌物种的CRISPR-Cas系统可以同时独立运行，因为它们具有不同的单导rna （sgRNAs）或crispr - rna （crrna）以及原间隔器邻近基序（PAMs）。此外，我们假设斑马鱼中的CRISPR-Cas活性可以通过抑制性抗crispr （Acr）蛋白的表达来调节。在这里，我们使用一种简单的诱变方法来证明来自化脓性链球菌（SpyCas9）、金黄色链球菌（SauCas9）、毛螺科细菌（LbaCas12a，以前称为LbCpf1）的CRISPR-Cas系统是能够在斑马鱼中同时工作的正交系统。来自酸胺球菌sp. （AspCas12a，以前称为AsCpf1）和脑膜炎奈瑟菌（Nme2Cas9）的CRISPR系统在胚胎中也有活性。我们使用三种CRISPR系统实现了多通道CRISPR记录，并表明与以前的方法相比，LbaCas12a可能提供更高的信息密度。我们还证明了II型Acrs（抗crispr）是斑马鱼中SpyCas9的有效抑制剂。我们的研究结果表明，至少有五种CRISPR-Cas系统和两种抗crispr蛋白在斑马鱼胚胎中起作用。这些正交的CRISPR-Cas系统和Acr蛋白将为动物基因组编辑和遗传记录的时空控制提供组合和交叉策略。
The CRISPR-Cas universe continues to expand. The type II CRISPR-Cas system from Streptococcus pyogenes (SpyCas9) is the most widely used for genome editing due to its high efficiency in cells and organisms. However, concentrating on a single CRISPR-Cas system imposes limits on target selection and multiplexed genome engineering. We hypothesized that CRISPR-Cas systems originating from different bacterial species could operate simultaneously and independently due to their distinct single-guide RNAs (sgRNAs) or CRISPR-RNAs (crRNAs), and protospacer adjacent motifs (PAMs). Additionally, we hypothesized that CRISPR-Cas activity in zebrafish could be regulated through the expression of inhibitory anti-CRISPR (Acr) proteins. Here, we use a simple mutagenesis approach to demonstrate that CRISPR-Cas systems from Streptococcus pyogenes (SpyCas9), Streptococcus aureus (SauCas9), Lachnospiraceae bacterium (LbaCas12a, previously known as LbCpf1), are orthogonal systems capable of operating simultaneously in zebrafish. CRISPR systems from Acidaminococcus sp. (AspCas12a, previously known as AsCpf1) and Neisseria meningitidis (Nme2Cas9) were also active in embryos. We implemented multichannel CRISPR recording using three CRISPR systems and show that LbaCas12a may provide superior information density compared to previous methods. We also demonstrate that type II Acrs (anti-CRISPRs) are effective inhibitors of SpyCas9 in zebrafish. Our results indicate that at least five CRISPR-Cas systems and two anti-CRISPR proteins are functional in zebrafish embryos. These orthogonal CRISPR-Cas systems and Acr proteins will enable combinatorial and intersectional strategies for spatiotemporal control of genome editing and genetic recording in animals.