Expansion of CRISPR Targeting Sites Using an Integrated Gene-Editing System in Apis mellifera.
Expansion of CRISPR Targeting Sites Using an Integrated Gene-Editing System in Apis mellifera.
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使用整合的基因编辑系统在意大利蜜蜂中扩展CRISPR靶向位点。
DOI：
10.3390/insects12100954
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发表时间：
2021-10-19
期刊：
影响因子：
3
通讯作者：
Nie H
中科院分区：
文献类型：
--
作者：
Liang L;Li Z;Li Q;Wang X;Su S;Nie H
CRISPR/Cas9是一种多功能的基因操作工具，已被用于蜜蜂的靶向基因组工程。然而，到目前为止，只有能够识别NGG的SpCas 9被证明可以操纵A.意大利蜜蜂，将可编辑范围限制为包含NGG的基因座。在目前的研究中，为了评估利用Cpf 1，SpCas 9和SaCas 9的潜在扩展，我们用生物信息学方法预测了整个蜜蜂基因组中靶向位点的分布和数量。生物信息学分析结果表明，A.通过整合的CRISPR系统，可以显著增加意大利产蜜蜂的数量。此外，我们测量了这些新CRISPR酶在A.发现SaCas 9和Cpf 1都能诱导意大利蜜蜂基因组的变异。尽管Cpf 1的诱变率相对较低，SaCas 9的编辑不稳定。据我们所知，我们的研究提供了第一个证据，证明SaCas 9和Cpf 1可以有效地介导基因组序列突变，从而扩大了A.意利费拉。整合的CRISPR系统可能会促进A. mellifera和其他昆虫。CRISPR/Cas9是一种主要的基因编辑工具，已被用于分析意大利蜜蜂中的基因功能。然而，只有含有NGG PAM的基因组区域可以在A.这严重阻碍了CRISPR技术在蜜蜂中的应用。在这项研究中，我们对NGG，TTN和NNGRRT的全基因组靶向位点进行了生物信息学分析，以确定SpCas 9，SaCas 9，Cpf 1的潜在扩展，发现通过整合基因操作系统可以显着扩展CRISPR编辑系统的靶向谱。同时，将不同新型基因编辑系统的单向导RNA（sgRNA）/crRNA和相应的CRISPR蛋白共注射到蜜蜂胚胎中，并在A.意利费拉。测序数据显示SaCas 9和Cpf 1都能够介导A.尽管Cpf 1的诱变率相对较低，SaCas 9的编辑不稳定。据我们所知，我们的结果首次证明了SaCas 9和Cpf 1可以诱导基因组序列改变，这将编辑范围扩展到TTN和NNGRRT的目标，并使基于CRISPR的基因组研究在更广泛的范围内在A.意利费拉。
CRISPR/Cas9, a versatile gene manipulation tool, has been harnessed for targeted genome engineering in honeybees. However, until now, only SpCas9 that enables NGG recognition has been shown to manipulate the genome in A. mellifera, limiting the editable range to the NGG-included loci. In the current study, to evaluate the potential expansion when utilising Cpf1, SpCas9 and SaCas9, we predicted the distribution and number of targeting sites throughout the whole honeybee genome with a bioinformatic approach. The results of bioinformatics analysis suggest that the number of accessible targeting sites in A. mellifera could be significantly increased via the integrated CRISPR system. In addition, we measured the cleavage activity of these new CRISPR enzymes in A. mellifera, and it was found that both SaCas9 and Cpf1 can induce genome alternation in A. mellifera, albeit with relatively lower mutagenesis rates for Cpf1 and unstable editing for SaCas9. To our knowledge, our study provides the first evidence that SaCas9 and Cpf1 can efficiently mediate genome sequence mutation, thereby expanding the targetable spectrum in A. mellifera. The integrated CRISPR system will probably boost both fundamental studies and applied researches in A. mellifera and perhaps other insects. CRISPR/Cas9, a predominant gene-editing tool, has been utilised to dissect the gene function in Apis mellifera. However, only the genomic region containing NGG PAM could be recognised and edited in A. mellifera, seriously hampering the application of CRISPR technology in honeybees. In this study, we carried out the bioinformatics analysis for genome-wide targeting sites of NGG, TTN, and NNGRRT to determine the potential expansion of the SpCas9, SaCas9, Cpf1, and it was found that the targetable spectrum of the CRISPR editing system could be markedly extended via the integrated gene manipulation system. Meanwhile, the single guide RNA (sgRNA)/crRNA of different novel gene editing systems and the corresponding CRISPR proteins were co-injected into honeybee embryos, and their feasibility was tested in A. mellifera. The sequencing data revealed that both SaCas9 and Cpf1 are capable of mediating mutation in A. mellifera, albeit with relatively lower mutagenesis rates for Cpf1 and unstable editing for SaCas9. To our knowledge, our results provide the first demonstration that SaCas9 and Cpf1 can function to induce genome sequence alternation, which extended the editing scope to the targets with TTN and NNGRRT and enabled CRISPR-based genome research in a broader range in A. mellifera.
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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
影响因子：
46.9
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46.9
作者：
Kleinstiver BP;Tsai SQ;Prew MS;Nguyen NT;Welch MM;Lopez JM;McCaw ZR;Aryee MJ;Joung JK
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Joung JK
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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