背景:目前正在评估种群抑制基因驱动,包括通过环境风险评估(ERA),用于疟疾病媒控制。一个这样的基因驱动涉及编码(i)hCas9内切核酸酶的dsxFCRISPRh转基因,(ii)靶向doublesex基因座的T1指导RNA(gRNA),和(iii)DsRed荧光标记蛋白,在转基因蚊子(GMM)。问题制定,ERA的第一阶段,对于dsxFCRISPRh的环境释放,先前确定了可能对环境或健康造成的九种潜在危害,应表达的转基因产物引起变应原性或毒性。
方法:hCas9和DsRed的氨基酸序列与来自NCBI的毒素或过敏原的氨基酸序列进行了询问,UniProt,比较和AllergenOnline生物信息学数据库和gRNA与miRBase数据库中的microRNA进行了比较,以了解对与毒性或过敏原性相关的基因表达的潜在影响。PubMed还搜索了Cas9或DsRed的毒性或过敏原性的任何证据,或这些产品最初来源的供体生物。
结果:虽然Cas9核酸酶活性在体外对某些细胞类型具有毒性,并且发现hCas9与原核细胞毒素VapC具有同源性,以前的研究没有证据表明hCas9对人类和其他动物的毒性风险.尽管hCas9确实含有在胶乳变应原Hevb9中发现的8聚体表位,但hCas9的完整氨基酸序列与任何已知变应原不同源。结合文献中缺乏Cas9致敏性的证据,这表明hCas9对人类的致敏性风险可以忽略不计.在来自按蚊或人的gRNA和microRNA之间没有发现匹配。此外,来自环境释放的dsxFCRISPRh转基因蛋白的潜在暴露被评估为可忽略不计。
结论:生物信息学和文献评估没有发现令人信服的证据表明dsxFCRISPRh表达的转基因产品是过敏原或毒素,这表明,用于控制疟疾病媒的这种种群抑制基因的环境释放不应导致人类或动物的变应原性或毒性增加。这些结果还应该为正在开发的用于CRISPR-Cas9的载体控制和体内临床应用的其他GMM的评估提供信息。
BACKGROUND: Population suppression gene drive is currently being evaluated, including via environmental risk assessment (ERA), for malaria vector control. One such gene drive involves the dsxFCRISPRh transgene encoding (i) hCas9 endonuclease, (ii) T1 guide RNA (gRNA) targeting the doublesex locus, and (iii)
DsRed fluorescent marker protein, in genetically-modified mosquitoes (GMMs). Problem formulation, the first stage of ERA, for environmental releases of dsxFCRISPRh previously identified nine potential harms to the environment or health that could occur, should expressed products of the transgene cause allergenicity or toxicity.
METHODS: Amino acid sequences of hCas9 and
DsRed were interrogated against those of toxins or allergens from NCBI, UniProt, COMPARE and AllergenOnline bioinformatic databases and the gRNA was compared with microRNAs from the miRBase database for potential impacts on gene expression associated with toxicity or allergenicity. PubMed was also searched for any evidence of toxicity or allergenicity of Cas9 or
DsRed, or of the donor organisms from which these products were originally derived.
RESULTS: While Cas9 nuclease activity can be toxic to some cell types in vitro and hCas9 was found to share homology with the prokaryotic toxin VapC, there was no evidence from previous studies of a risk of toxicity to humans and other animals from hCas9. Although hCas9 did contain an 8-mer epitope found in the latex allergen Hev b 9, the full amino acid sequence of hCas9 was not homologous to any known allergens. Combined with a lack of evidence in the literature of Cas9 allergenicity, this indicated negligible risk to humans of allergenicity from hCas9. No matches were found between the gRNA and microRNAs from either Anopheles or humans. Moreover, potential exposure to dsxFCRISPRh transgenic proteins from environmental releases was assessed as negligible.
CONCLUSIONS: Bioinformatic and literature assessments found no convincing evidence to suggest that transgenic products expressed from dsxFCRISPRh were allergens or toxins, indicating that environmental releases of this population suppression gene drive for malaria vector control should not result in any increased allergenicity or toxicity in humans or animals. These results should also inform evaluations of other GMMs being developed for vector control and in vivo clinical applications of CRISPR-Cas9.