Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs.
Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs.
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DOI：
10.1073/pnas.1805278115
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发表时间：
2018-10-02
影响因子：
11.1
通讯作者：
Hay BA
中科院分区：
文献类型：
--
作者：
Oberhofer G;Ivy T;Hay BA
基于同源内切酶基因(HEG)的基因驱动可以在以生存或生育所需的基因为靶点时带来种群抑制。然而，这些策略很容易失败，因为这些策略产生了抵抗卵裂的等位基因，但保留了野生型基因的功能。我们发现，通过使用设计用于在四个靶点切割基因的引导RNA，可以防止抗性等位基因的产生。然而，寻的速度是适度的，黑格人在寻的过程中并不稳定。此外，使用在雌性生殖系中活跃的启动子会导致HEG水平的残留，这会损害携带HEG的杂合子的生存能力或生育能力，从而阻止驱动力。我们提出了在下一代HEG系统中帮助克服这些问题的策略。一种对种群抑制特别感兴趣的基因驱动方法利用归巢内切酶基因(HEGS)，其中在生殖系中将位置特异的核酸酶编码盒复制到目标基因中，该目标基因的功能丧失会导致纯合但不是杂合的后代丧失生存能力或生育能力。在果蝇和蚊子的早期研究中，使用了由Cas9和单一引导RNA(GRNA)组成的Hegs，它们一起针对特定的基因进行切割。观察到了归巢，但免疫切割的抗性等位基因在保留野生型基因功能的同时，也通过非同源末端连接产生。这样的等位基因可以防止驱动力和种群抑制。针对一个基因在多个位置进行切割，已被认为是防止出现抗性等位基因的一种策略。为了验证这一假设，我们在果蝇中产生了两个针对胚胎存活或生育所需基因的抑制Hegs，使用由CRISPR/Cas9和gRNA组成的HEG，这些基因被设计成在四个位置切割每个基因。靶基因的裂解率很高，gRNAs的多重作用阻碍了抗性等位基因的形成。然而，生殖系归巢的比率不高，而且HEG盒在归巢事件期间不稳定，导致缺乏显性标记基因gRNAs或Cas9的Hegs频繁的部分复制。最后，在驾驶实验中，由于母体携带Cas9/gRNA复合体活性而在后代中诱导了高适应负荷，Hegs未能传播。提出了可供选择的设计原则，以缓解未来基因驱动工程中的这些问题。
Homing endonuclease gene (HEG)-based gene drive can bring about population suppression when genes required for viability or fertility are targeted. However, these strategies are vulnerable to failure through mechanisms that create alleles resistant to cleavage but that retain wild-type gene function. We show that resistance allele creation can be prevented through the use of guide RNAs designed to cleave a gene at four target sites. However, homing rates were modest, and the HEGs were unstable during homing. In addition, use of a promoter active in the female germline resulted in levels of HEG carryover that compromised the viability or fertility of HEG-bearing heterozygotes, thereby preventing drive. We propose strategies that can help to overcome these problems in next-generation HEG systems. A gene drive method of particular interest for population suppression utilizes homing endonuclease genes (HEGs), wherein a site-specific, nuclease-encoding cassette is copied, in the germline, into a target gene whose loss of function results in loss of viability or fertility in homozygous, but not heterozygous, progeny. Earlier work in Drosophila and mosquitoes utilized HEGs consisting of Cas9 and a single guide RNA (gRNA) that together target a specific gene for cleavage. Homing was observed, but resistant alleles immune to cleavage, while retaining wild-type gene function, were also created through nonhomologous end joining. Such alleles prevent drive and population suppression. Targeting a gene for cleavage at multiple positions has been suggested as a strategy to prevent the appearance of resistant alleles. To test this hypothesis, we generated two suppression HEGs in Drosophila melanogaster targeting genes required for embryonic viability or fertility, using a HEG consisting of CRISPR/Cas9 and gRNAs designed to cleave each gene at four positions. Rates of target locus cleavage were very high, and multiplexing of gRNAs prevented resistant allele formation. However, germline homing rates were modest, and the HEG cassette was unstable during homing events, resulting in frequent partial copying of HEGs that lacked gRNAs, a dominant marker gene, or Cas9. Finally, in drive experiments, the HEGs failed to spread due to the high fitness load induced in offspring as a result of maternal carryover of Cas9/gRNA complex activity. Alternative design principles are proposed that may mitigate these problems in future gene drive engineering.
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CRIS/CAS9活性的生物物理模型，用于基因组编辑和基因调节的合理设计。
影响因子：
4.3
作者：
Farasat I;Salis HM
通讯作者：
Salis HM
DOI：
10.1098/rsif.2013.1071
发表时间：
2014-04-06
期刊：
Journal of the Royal Society, Interface
影响因子：
--
作者：
Alphey N;Bonsall MB
通讯作者：
Bonsall MB
影响因子：
12.3
作者：
Dang Y;Jia G;Choi J;Ma H;Anaya E;Ye C;Shankar P;Wu H
通讯作者：
Wu H
DOI：
10.1126/science.aaa5945
发表时间：
2015-04-24
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Gantz VM;Bier E
通讯作者：
Bier E
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE