Pronuclear Microinjection during S-Phase Increases the Efficiency of CRISPR-Cas9-Assisted Knockin of Large DNA Donors in Mouse Zygotes
Pronuclear Microinjection during S-Phase Increases the Efficiency of CRISPR-Cas9-Assisted Knockin of Large DNA Donors in Mouse Zygotes
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DOI：
10.1016/j.celrep.2020.107653
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发表时间：
2020-05-19
期刊：
影响因子：
8.8
通讯作者：
Kiyonari, Hiroshi
中科院分区：
文献类型：
--
作者：
Abe, Takaya;Inoue, Ken-ichi;Kiyonari, Hiroshi
在受精卵中的CRISPR-Cas9辅助敲入（KI）中，剩余的挑战是常规地实现大（内切酶大小的）DNA元件的高效KI。在这里，我们专注于原核注射的时机，并建立了一个可靠的同源重组（HR）为基础的方法，以产生大的Kls在受精卵相比，其他两种类型的KI策略，涉及不同的DNA修复途径。在ROSA 26基因座处，通过使用基于HR的方法在S期用CRISPR RNA（crRNA）、反式激活crRNA（tracrRNA）和Cas9蛋白质进行原核注射产生最有效和准确的Kls（高达70%）。这种方法对于在其他基因座产生大的KI等位基因通常也是有效的。我们进一步应用我们的方法，通过顺序注射到两个原核中来有效地获得双等位基因ROSA 26 Kls。我们的研究结果表明，将基因组编辑组分和供体DNA递送到S期合子中对于大DNA元件的有效KI至关重要。
In CRISPR-Cas9-assisted knockin (KI) in zygotes, a remaining challenge is routinely achieving high-efficiency KI of large (kilobase-sized) DNA elements. Here, we focus on the timing of pronuclear injection and establish a reliable homologous recombination (HR)-based method to generate large Kls in zygotes compared with two other types of KI strategies involving distinct DNA repair pathways. At the ROSA26 locus, pronuclear injection with CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9 protein at the S phase by using the HR-based method yields the most efficient and accurate Kls (up to 70%). This approach is also generally effective for generating large KI alleles at other gene loci. We further apply our method to efficiently obtain biallelic ROSA26 Kls by sequential injection into both pronuclei. Our results suggest that delivery of genome editing components and donor DNA into S-phase zygotes is critical for efficient KI of large DNA elements.