CN114292830B - 蛋白SaCas9的抗原表位及其在基因编辑中的应用 - Google Patents
蛋白SaCas9的抗原表位及其在基因编辑中的应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，提供了一种突变的Cas9蛋白，其具有剪切基因的活性并且其突变是能够降低免疫原性的抗原表位，选自A144G，E146G，E147G，D161G，G162A，R269G，D270A，E271G，N272G，E273G，P294A，T295A，D309A，P321A，E322G，A337G，R338G，T369A，E378G或短肽200‑208 TYIDLLETR。本发明还包括SaCas9蛋白的抗原表位及其在基因编辑方面的应用。本发明的优势抗原表位对SaCas9蛋白切割功能活性无影响，对Cas9蛋白的去免疫化改造能改善CRISPR‑Cas9***可能诱发的人体免疫反应问题。
Description
技术领域
本发明属于生物技术领域，涉及SaCas9蛋白的抗原表位，包括SaCas9蛋白的突变体及其在基因编辑方面的应用。
背景技术
CRISPR-Cas9作为一种新型基因编辑工具，因其高效性和简便性而广泛应用于实验室研究中，并逐渐应用于多种人类疾病的基因治疗中。CRISPR(Clustered RegularlyInterspaced Short Palindromic Repeats)中文全称是规律间隔成簇短回文重复序列，Cas是CRISPR相关蛋白的简称，CRISPR和Cas构成CRISPR–Cas***。CRISPR–Cas***中，Cas9能够靶向地切除在细菌内的外源DNA，而不会切除自身DNA。CRISPR技术依靠一种利用引导性RNA分子将其导向目标DNA、Cas9按照预定方式编辑DNA，可以纠正导致疾病的基因错误，消除导致疾病的微生物，治疗复杂疾病，培育更健康的新型食物，根除地球最危险的害虫乃至复活珍惜物种。
Cas9蛋白作为介导CRISPR-Cas9***行使功能的重要组成部分，已有多种同源蛋白被发现并应用。其中Staphylococcus aureus Cas9(SaCas9)因其较小的分子量、较高的基因编辑效率和较低的脱靶风险，尤其易于被重组腺相关病毒(AAV)载体包装，而将成为临床基因治疗的有力工具。
然而，在这项技术应用于临床基因治疗的过程中，除了需要避免SaCas9潜在的脱靶效应外，也要克服人体可能产生的针对SaCas9蛋白的免疫反应。研究发现人体预先存在着Cas9蛋白特异的免疫反应，包括先天免疫和适应性免疫，这些免疫反应的存在可能会使治疗效果降低，安全性风险提高。历史上，基因治疗曾因为一位患者因载体引发***性炎症致死而陷入停滞。宾大Carl.H.June团队发现，Ex vivo基因治疗I期临床检测到患者Cas9蛋白的免疫原性极低。但是与ex vivo不同，in vivo途径或者表观遗传疗法需要体内长期表达Cas9，例如，使用AAV递送CRISPR-Cas9***到小鼠肝脏，会导致CD8+T细胞在肝脏聚集、导致肝损伤，这表明免疫原性为题将会成为CRISPR-Cas9临床应用的巨大障碍。
因此，利用蛋白质工程手段改造SaCas9蛋白，获得功能活性不受影响的低免疫原性突变体，可以有效地解决Cas9蛋白诱发的人体免疫反应问题，进而提高CRISPR-Cas9靶向基因治疗的安全性和有效性，加快基因编辑技术向临床转化的进程。
发明内容
本发明要解决的技术问题是在不改变Cas9蛋白基因剪切活性的前提下降低其免疫原性，从而提高CRISPR-Cas9在基因治疗临床应用中的安全性和有效性。
针对人体预先存在的对SaCas9蛋白的免疫反应可能影响CRISPR Cas9***在临床应用上的安全性和有效性这一问题，本发明首先采用ELISA和ELISpot方法，分别检测了123位中国健康个体血清中SaCas9蛋白特异的IgG抗体水平，以及10个中国健康个体PBMCs中SaCas9蛋白特异的T细胞激活水平，发现约4.8％的个体呈SaCas9特异抗体水平阳性，约70％的工体T细胞可被SaCas9激活。但是获得有效的抗原表位很困难，目前科学家们通常采用多个抗原表位联合使免疫原性降低，但是能达到的最好的效果约是免疫原性下降50％。本发明的研究中也发现筛选过程工作量大，而且不确定因素很多。首先收集阳性的T细胞和相关B细胞用于辅助筛选抗原表位就需要寻找合适的方法并设定合理的阈值，影响免疫原性因素很多，因此筛选的方法和标准也需要反复验证。其次是获得第一阶段突变后需要设计引物、装载体、验证突变，这其中的工作量很大。再次也是不确定的原因之一，在Cas9蛋白纯化过程中，因为使用前要去除内毒素、而且需要的蛋白数量很大，而该蛋白本身分子量很大，无论是挂柱还是纯化过程中都可能大量损失甚至不能获得纯化的蛋白。经过反复的摸索，本发明终于成功获得了符合预期的抗原表位和突变蛋白，简要的研究过程如下：
本发明通过生物信息学分析预测了SaCas9蛋白的B细胞抗原表位，以及针对中国人群中最常见HLA分型的CD8+T细胞抗原表位。随后依据预测出的B细胞抗原表位，定向突变SaCas9蛋白，排除对其功能活性的影响后，使用SaCas9抗体水平阳性的健康人供体血清检测分析突变型SaCas9的抗原抗体反应水平，筛选并确认B细胞优势抗原表位，得到使人体内预先存在的SaCas9特异的体液免疫反应水平降低且功能活性不受影响的改良型SaCas9蛋白；依据预测出的CD8+T细胞表位合成多肽,使用SaCas9特异激活T细胞阳性的供体PBMCs检测分析候选表位多肽的T细胞激活水平，鉴定出SaCas9蛋白针对中国人群的CD8+T细胞优势抗原表位。本发明在上述基础上完成。
一方面，本发明提供了一种突变的Cas9蛋白，所述的Cas9蛋白是SaCas9蛋白，源自Staphylococcus aureus；或者，所述突变的Cas9蛋白源自SEQ ID NO 1：
AKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG(SEQID NO 1)。
所述的Cas9蛋白具有剪切基因的活性并且其突变选自但不限于：A144G，E146G，E147G，D161G，G162A，D270A，E271G，E273G，P294A，T295A，D309A，P321A，E322G，A337G，R338G，T369A，E378G。
较好的，所述的突变选自E271G，P321A，R338G。
本发明通过将B细胞优势抗原表位定向突变为甘氨酸，得到了一种改良型SaCas9蛋白，可以显著降低人体内预先存在的SaCas9特异的体液免疫反应水平，且切割功能活性不受影响。
本发明的SaCas9蛋白B细胞抗原表位选自：A144G，E146G，E147G，D161G，G162A，D270A，E271G，E273G，P294A，T295A，D309A，P321A，E322G，A337G，R338G，T369A，E378G。同时，本发明提供了一种SaCas9蛋白的CD8+T细胞抗原表位，位于200-208的氨基酸TYIDLLETR(SEQ ID NO 3)。
相应的，本发明提供了一种核酸，所述的核酸是突变的Cas9蛋白或者其抗原表位的编码序列。
另一方面，本发明提供了上述的突变的Cas9蛋白或者其抗原表位，以及其编码序列的制备方法。
上述的突变的Cas9蛋白或者其抗原表位，以及其编码序列，可以根据已知的序列人工合成。或者，按照冷泉港的《分子克隆》操作，也可以在源自Staphylococcus aureus的SaCas9蛋白基础上通过筛选获得，包括以下步骤：
A.通过计算机辅助筛选SaCas9蛋白中具备免疫原性但不位于其基因剪切功能位点的抗原表位；
B.根据筛选到的抗原表位构建突变的SaCas9蛋白或合成肽段；
C.使用SaCas9特异抗体阳性的供体血清对突变的SaCas9蛋白进行抗原抗体反应检测并剔除假阳性位点；或者使用SaCas9特异激活T细胞阳性的供体PBMCs检测分析并剔除T细胞激活水平达不到阈值的候选表位；
D.通过基因剪切实验验证突变的SaCas9蛋白，并剔除失去基因剪切作用的SaCas9蛋白。
其中，通过计算机辅助筛选SaCas9蛋白中具备免疫原性但不位于其基因剪切功能位点的抗原表位是指，
对于B细胞表位，包括：
根据SaCas9蛋白的氨基酸序列和晶体结构模型预测其B细胞抗原表位，使用DISCOTOPE2.0与IEDB ELLIPRO软件基于连续表位和不连续表位对每个氨基酸残基进行突出指数评分，以PI＝0.5、残基距离作为阈值，筛选出符合要求的线性表位和构象表位，并获得第一阶段B细胞抗原表位；
对SaCas9蛋白所有氨基酸残基进行亲水性参数、表面可及性参数、抗原性参数和柔韧性参数进行综合预测分析，以亲水性参数≥0，表面可及性参数≥1，抗原性参数≥1作为筛选标准，在所有残基中得到B细胞抗原表位的优势区段，作为第二阶段B细胞抗原表位；
将第一阶段B细胞抗原表位和第二阶段B细胞抗原表位结果进行汇总，选取重合的135个氨基酸位点作为SaCas9蛋白B细胞抗原表位最终的预测结果，即第三阶段B细胞抗原表位；
根据第三阶段B细胞抗原表位，排除功能结构域上的位点后，将其在SaCas9蛋白上分别突变为甘氨酸或丙氨酸，同时构建携带指示标记的切割活性检测***，在该检测***上筛选获得不影响切割活性的突变的SaCas9蛋白，并将相应的17个氨基酸位点作为第四阶段B细胞抗原表位；可以采用传统的切割活性检测***，本发明的快速切割活性检测***需要首先构建共表达荧光素酶基因和嘌呤抗性基因的载体，将其作为目的质粒包装慢病毒，感染Hela细胞系，加入嘌呤霉素多次筛选，得到稳定的表达荧光素酶的Hela-Luc。然后以荧光素酶为靶序列设计sgRNA，在Hela-Luc细胞系上筛选得到SaCas9蛋白切割活性检测模型。
获得切割活性不受影响的第四阶段B细胞抗原表位单氨基酸突变的SaCas9蛋白，作为抗原检测其与SaCas9特异抗体阳性的供体血清的抗原抗体反应水平，抗原性下降大于等于50％的B细胞抗原表位作为第五阶段B细胞抗原表位。
对于T细胞表位，获得的方法包括：
为了获得第一阶段CD8+T细胞抗原表位，需要收集细胞免疫反应阳性供体。具体方法为：采用ELISA和ELISpot方法，分别检测了123位中国健康个体血清中SaCas9蛋白特异的IgG抗体水平，以及10个中国健康个体PBMCs中SaCas9蛋白特异的T细胞激活水平，通过斑点计数和计算SFC，约70％的工体T细胞可被SaCas9激活，即视为细胞免疫反应阳性供体。
首先，使用IEDB HLA-I软件根据SaCas9蛋白的氨基酸序列预测SaCas9蛋白的CD8+T细胞抗原表位，预测对象分别选择在中国人群中最常见的3种HLA-I型等位基因：HLA-A*11:01(41.8％)、HLA-A*24:02(31.8％)和HLA-A*02:01(24.1％)，针对九肽进行预测，对3种HLA-I分子均选择了结合系数评分前30％的九肽为结合表位肽，选取结合表位肽中重合的50条多肽作为第一阶段CD8+T细胞抗原表位；
其次，使用GalaxyPepDock分子对接模拟软件进行HLA-I分子与50条肽段的对接模拟，分别分析三种HLA-I分子与九肽结合的情况，计算结合自由能，并排除位于基因剪切功能结构域的多肽，得到了预测结果最优的10条多肽作为第二阶段CD8+T细胞抗原表位；
然后，使用SaCas9蛋白特异激活T细胞阳性的供体PBMCs，通过ELISpot方法检测了第二阶段CD8+T细胞抗原表位混合肽对阳性供体PBMCs中T细胞的激活水平，获得能够对T细胞产生激活作用的第三阶段CD8+T细胞抗原表位；
最后，对第三阶段CD8+T细胞抗原表位单肽段分别进行T细胞激活检测，最终得到SaCas9蛋白适用于中国人群队列的CD8+T细胞优势抗原表位，即第四阶段CD8+T细胞抗原表位。在本发明的一个优选例中，筛选到的第四阶段CD8+T细胞抗原表位的氨基酸序列为TYIDLLETR(SEQ ID NO 3)。
再一方面，本发明还提供了突变的Cas9蛋白或SaCas9蛋白的抗原表位在基因编辑中的应用。
本发明提供了SaCas9蛋白的抗原表位在Cas9蛋白去免疫化改造中的应用。
较好的，所述的应用是突变的Cas9蛋白或SaCas9蛋白的抗原表位在制备基因编辑试剂或者药物中的应用；或者，所述突变的蛋白或抗原表位在进行Cas9蛋白的去免疫化改造中的应用；或者，所述突变的蛋白或抗原表位在CRISPR-Cas9***的基因编辑治疗中的应用。
较好的，所述的应用包括以下步骤：
将突变的Cas9蛋白的编码序列与表达载体重组；和/或将突变的Cas9蛋白替代未突变的Cas9蛋白，与CRISPR-Cas9***其他组成部分共同应用于基因编辑。
本发明通过生物信息学分析与免疫学方法验证相结合的手段，使用健康人的SaCas9抗体阳性血清和SaCas9特异激活T细胞阳性PBMCs筛选验证预测的候选抗原表位中，SaCas9蛋白的B细胞优势抗原表位和针对中国人群最常见HLA等位基因型的CD8+T细胞优势抗原表位。本发明使用了三年多时间，在筛选SaCas9蛋白的B细胞优势抗原表位和针对中国人群最常见HLA等位基因型的CD8+T细胞优势抗原表位的过程中，构建了大量的突变体，并收集了足够多的供体样本以筛选SaCas9特异适应免疫反应阳性的供体样本，通过多种方法、多重检验，衡量了多个参数和阈值，终于获得了符合预期的SaCas9蛋白优势抗原表位。尤其在对B细胞优势抗原表位定向突变后，将之重组到CRISPR-Cas9***中以替代原有的Cas9蛋白。进一步通过功能活性检验，确定该抗原表位的优化不会对SaCas9蛋白的切割功能活性产生影响，对未来Cas9蛋白的去免疫化改造和解决CRISPR-Cas9***可能诱发人体的免疫反应问题具有重大意义。
附图说明
图1：预测的SaCas9蛋白B细胞抗原表位三维结构示意图。
根据SaCas9蛋白的氨基酸序列和晶体结构模型预测其B细胞抗原表位，使用DISCOTOPE2.0与IEDB ELLIPRO软件基于连续表位和不连续表位对每个氨基酸残基进行突出指数评分，以PI＝0.5、残基距离作为阈值，筛选出符合要求的线性表位和构象表位，并获得第一阶段B细胞抗原表位；对SaCas9蛋白所有氨基酸残基进行亲水性参数、表面可及性参数、抗原性参数和柔韧性参数进行综合预测分析，以亲水性参数≥0，表面可及性参数≥1，抗原性参数≥1作为筛选标准，在所有残基中得到B细胞抗原表位的优势区段，作为第二阶段B细胞抗原表位；将第一阶段B细胞抗原表位和第二阶段B细胞抗原表位结果进行汇总，选取重合的135个氨基酸位点作为SaCas9蛋白B细胞抗原表位最终的预测结果，绘制成预测的SaCas9蛋白B细胞抗原表位三维结构示意图。
图2：候选B细胞抗原表位突变SaCas9蛋白切割活性的快速检测结果图。
使用荧光素酶检测体系对野生型和候选B细胞抗原表位单氨基酸突变的23个突变型SaCas9蛋白进行了切割活性的快速检测。如图2所示，在A144G，E146G，E147G，R157G，D161G，G162A，D270A，E273G，P294A，T295A，D309A，P321A，E322G，A337G，R338G，T369A，E378G，D79G，R269G，E271G，N272G，E308G,E340G等23个突变中发现有6个位点D79G，R269G，E271G，N272G，E308G,E340G的氨基酸改变后会显著影响SaCas9蛋白的切割功能活性，其余17个突变型SaCas9蛋白的切割活性相比于野生型无显著差异，从而进行后续的抗原性验证。LentiRV2-Luc慢病毒载体的构建：荧光素酶基因与嘌呤霉素抗性基因以EF-1α为启动子，通过P2A相连。荧光素酶基因的Cas9蛋白切割靶点sgRNA共列举了3条。
图3：SaCas9蛋白B细胞优势抗原表位的筛选验证图。
使用从中国人群队列中筛选得到的SaCas9蛋白特异IgG抗体水平阳性的血清作为检测抗体，通过ELISA的方法分别定量检测了各个突变型和野生型SaCas9蛋白与抗体阳性供体血清的抗原抗体反应水平，计算各个SaCas9蛋白突变体以野生型为基准的相对抗原抗体反应水平，抗原性下降最显著的突变型SaCas9蛋白的氨基酸突变位点即为B细胞优势抗原表位。通常文献报道以免疫原性下降50％作为显著差异，E271G、P321A、R338G均能够使其免疫原性显著降低，而本发明中最好的位点R338G突变后的SaCas9蛋白抗原性下降约70％。
图4：改良型SaCas9蛋白功能活性的验证图。
其中凝胶电泳图为野生型和改良型SaCas9蛋白切割目标基因(荧光素酶基因)后，对靶点处的序列进行T7E1酶切检测的结果。阴性对照呈800bp单条带，成功切割后的目标基因片段呈530bp与270bp双条带，通过计算条带灰度，可以得到各SaCas9蛋白对目标基因的切割效率，汇总至表中。测序结果为野生型和改良型SaCas9蛋白切割目标基因后，对靶点处的序列进行测序的切割阳性结果示意图。对每个实验组切割后的基因片段，均随机选取10-20个克隆进行测序，计算切割阳性的克隆数，从而得到切割效率，汇总至表中。发现R338G改良型SaCas9蛋白的切割功能活性与野生型相比无显著差异，R157G突变型SaCas9作为前期筛选得到的功能活性影响位点，在本实验中作为方法学对照组，证明了方法的可行性与稳定性。
图5：预测的SaCas9蛋白针对3种中国人群最常见HLA等位基因型的CD8+T细胞抗原表位多肽及其预测参数。
其中，根据SaCas9蛋白的氨基酸序列，采用IEDB提供的MHC-I结合预测软件默认推荐的预测方法选项，基于SaCas9蛋白和HLA分子匹配最合适的九肽的预测方法。预测对象选择在中国人群中最常见的3种HLA-I型等位基因：HLA-A*11:01(41.8％)、HLA-A*24:02(31.8％)和HLA-A*02:01(24.1％)。选取重合的50条肽段作为对中国人群较为广谱的SaCas9蛋白候选CD8+T细胞抗原表位。同时，使用GalaxyPepDock分子对接模拟软件进行HLA分子与50条候选九肽的对接模拟，分别分析三种HLA分子与九肽结合的情况。进而使用AutoDock软件对GalaxyPepDock给出的对接结构进行进一步分析，利用AutoDock自带算法计算结合自由能，根据结合自由能高低，对预测结果的优先度进行初步评估。50条候选CD8+T细胞抗原表位多肽与3种HLA-I分子的结合自由能和IEDB结合系数如图5所展示。
图6：SaCas9蛋白CD8+T细胞优势抗原表位的筛选验证图。
综合考虑结合自由能和结合系数，并排除位于切割活性结构域的多肽，得到了预测结果最优的10条候选CD8+T细胞抗原表位多肽，进行合成。使用从中国人群队列中筛选得到的SaCas9蛋白特异激活T细胞阳性的供体PBMCs，通过ELISpot方法检测了5组混合肽对阳性供体PBMCs中T细胞的激活水平，发现3组和5组的多肽可检测到对T细胞的激活，其中3组激活水平更高。进而对两组共4条多肽通过ELISpot方法分别进行了T细胞激活检测，最终得到了SaCas9蛋白适用于中国人群队列的CD8+T细胞优势抗原表位，为SEQ ID NO 3所示的9肽。其中，SFC是斑点形成细胞数；PHA是植物血球凝集素，作为阳性对照；DMSO是二亚基甲酚，作为阴性对照。
具体实施方式
本发明属于生物技术领域，提供了SaCas9蛋白的抗原表位。本发明通过生物信息学分析与免疫学方法验证相结合的手段，利用健康人的SaCas9特异抗体阳性血清和SaCas9特异激活T细胞阳性PBMCs，获得了SaCas9蛋白B细胞优势抗原表位和针对中国人群最常见HLA等位基因型的CD8+T细胞优势抗原表位SEQ ID NO 3。同时通过将B细胞优势抗原表位定向突变，获得了使人体内预先存在的SaCas9特异的体液免疫反应降低且功能活性不受影响的改良型SaCas9蛋白SEQ ID NO 2：
AKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITAGKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG.(SEQ ID NO 2)。
本发明所提供的抗原表位为SaCas9蛋白的去免疫化研究和改造提供了前期研究基础，为解决CRISPR-Cas9***可能诱发人体产生免疫反应的问题提供了可能性。以下结合实施例对本发明作进一步的说明。
实施例1.SaCas9蛋白B细胞抗原表位的预测
首先从RCSB PDB蛋白数据库中检索得到了SaCas9蛋白的氨基酸序列和晶体结构模型(PDB：5AXW)，再将序列和结构模型输入到IEDB ELLIPRO和DISCOTOPE 2.0蛋白B细胞抗原表位在线预测平台中。
ELLIPRO基于连续(线性)表位和不连续(构象)表位对每个氨基酸残基进行评分。对每一个被预测到的表位进行PI(Protrusion Index，突出指数)评分，采用默认设置以PI＝0.5作为阈值，即突出指数高于50％的残基将被识别为表位；采用默认设置以残基距离作为阈值，即距离小于/>的残基可被识别为一个不连续表位，由此分别得到了441个可能的线性表位、499个可能的构象表位。
DiscoTope 2.0采用默认设置以-3.7作为阈值，其含义是25％的非表位残基可能作为误差被预测为部分表位，以及可保证表位残基中至少47％可被预测为部分表位，由此得到了596个可能的B细胞抗原表位。
使用DNAStar软件的Protean功能，对所有氨基酸残基进行亲水性参数(Kyte-Doolittle方案)、表面可及性参数(Emini方案)、抗原性参数(Jameson-Wolf方案)和柔韧性参数(Karplus-Schulz方案)的预测分析，以亲水性参数≥0，表面可及性参数≥1，抗原性参数≥1作为筛选标准，在所有残基中得到B细胞抗原表位的优势区段，综合分析这四种参数，得到了446个可能的B细胞抗原表位氨基酸残基。
将各软件的抗原表位预测结果进行汇总，选取重合的135个氨基酸位点作为SaCas9蛋白B细胞抗原表位最终的预测结果，绘制候选B细胞抗原表位的三维结构示意图(图1)。
实施例2.突变型SaCas9蛋白切割活性的快速检测
综合各预测软件的参数值，并排除切割功能结构域和已报道的功能位点的氨基酸残基，得到了预测结果最优的23个候选B细胞抗原表位氨基酸残基。通过点突变的方法对23个位点的氨基酸分别进行了突变，为了在改变其抗原性的基础上减少对蛋白其他性质的影响，将其全部突变为中性且基团最小的甘氨酸或丙氨酸。
使用预先构建的SaCas9蛋白切割活性快速检测体系，该体系为稳定表达荧光素酶的Hela细胞系，将待检测突变型SaCas9与靶向荧光素酶的sgRNA串联表达，以空质粒转染组为对照，对野生型和候选B细胞抗原表位单氨基酸突变的23个突变型SaCas9蛋白进行了切割活性的快速检测，如图2所示，发现有6个位点的氨基酸改变后会显著影响SaCas9蛋白的切割功能活性，其余17个突变型SaCas9蛋白的切割活性相比于野生型无显著差异，从而进行后续的抗原性验证。
实施例3.SaCas9蛋白B细胞抗原表位的验证
根据预测的SaCas9蛋白B细胞抗原表位候选结果，结合突变型SaCas9蛋白切割活性的快速检测结果，将17个切割活性不受影响的B细胞抗原表位单氨基酸突变SaCas9蛋白的表达质粒瞬时转染HEK293-17细胞系。转染后2d-3d裂解细胞提取蛋白，使用Anti-Flag-Magnetic Beads对细胞裂解后的全蛋白进行了IP纯化，最终得到了17个候选B细胞抗原表位单氨基酸突变SaCas9蛋白。
将17个SaCas9蛋白突变体和野生型SaCas9蛋白定量后，作为抗原等量包被到ELISA板中。如图3所示，使用从中国人群队列中筛选得到的SaCas9蛋白特异IgG抗体水平阳性的血清作为检测抗体，通过ELISA的方法分别定量检测了各个突变型和野生型SaCas9蛋白与抗体阳性血清的抗原抗体反应水平，将野生型SaCas9蛋白的抗原抗体反应水平设为1，通过归一法计算各个SaCas9蛋白突变体以野生型为基准的相对抗原抗体反应水平，抗原性下降最显著的突变型SaCas9蛋白的氨基酸突变位点即为优势抗原表位。其中第338位的B细胞优势抗原表位，R突变为G后的SaCas9蛋白抗原性下降约70％。
实施例4.改良型SaCas9蛋白功能活性的验证
使用T7E1酶切和测序方法验证B细胞优势抗原表位突变的改良型SaCas9蛋白的切割活性，以快速检测中切割活性降低的R157G突变SaCas9蛋白为方法学对照组，通过将等量的野生型和突变型SaCas9克隆与靶向荧光素酶基因的sgRNA共同转染Hela-Luc细胞系，提取转染后72h的细胞基因组。
对靶向基因进行巢式PCR，得到包含SaCas9切割靶点的基因片段，使用识别切割基因indel位点的T7核酸内切酶对该基因片段进行酶切，通过琼脂糖凝胶电泳结果判断野生型和突变型SaCas9蛋白对靶基因的切割水平。
同时将巢式PCR得到的基因片段构建到T载体上，转化后挑取10-20个单克隆对切割靶点处进行测序，最终通过计算电泳结果图中切割与未切割条带灰度的比例，以及测序Indel阳性克隆的比例，证明了B细胞优势抗原表位突变SaCas9蛋白的切割活性与野生型相比无显著差异。
实施例5.SaCas9蛋白CD8+T细胞抗原表位的预测
使用IEDB提供的MHC-I结合在线预测软件预测SaCas9蛋白的CD8+T细胞抗原表位。输入SaCas9蛋白的氨基酸序列，采用IEDB提供的MHC-I结合预测软件默认推荐的预测方法选项，基于SaCas9蛋白和HLA分子匹配最合适的预测方法。预测对象选择在中国人群中最常见的3种HLA-I型等位基因：HLA-A*11:01(41.8％)、HLA-A*24:02(31.8％)和HLA-A*02:01(24.1％)，针对九肽进行预测。选取重合的50条肽段作为对中国人群较为广谱的SaCas9蛋白候选CD8+T细胞抗原表位。
同时使用GalaxyPepDock分子对接模拟软件进行HLA分子与50条候选九肽的对接模拟，分别分析三种HLA分子与九肽结合的情况。进而使用AutoDock软件对GalaxyPepDock给出的对接结构进行进一步分析，利用AutoDock自带算法计算结合自由能，根据结合自由能高低，对预测结果的优先度进行初步评估。50条候选CD8+T细胞抗原表位多肽与3种HLA-I分子的结合自由能和IEDB结合系数如图5。
实施例6.SaCas9蛋白CD8+T细胞抗原表位的确定
综合考虑结合自由能和结合系数，并排除位于切割活性结构域的多肽，得到了预测结果最优的10条候选CD8+T细胞抗原表位多肽，进行合成。
如图6所示，使用从中国人群队列中筛选得到的SaCas9蛋白特异激活T细胞阳性的供体PBMCs，通过ELISpot方法检测了5组混合肽对阳性供体PBMCs中T细胞的激活水平，通过计算SFC(斑点形成细胞数)，发现3组和5组的多肽可检测到对T细胞的激活，其中3组的斑点形成细胞数更多，激活水平更高。进而对两组共4条多肽通过ELISpot方法分别进行了T细胞激活检测，最终得到多肽3-2，序列如SEQ ID NO 3(200-208)所示的SaCas9蛋白适用于中国人群队列的CD8+T细胞优势抗原表位。
上述相关说明以及对实施例的描述是为便于该技术领域的普通技术人员能理解和应用本发明。熟悉本领域技术的人员显然可以容易地对这些内容做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述相关说明以及对实施例的描述，本领域的技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
序列表
<110> 复旦大学
<120> 蛋白SaCas9的抗原表位及其在基因编辑中的应用
<141> 2021-12-31
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Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala
945 950 955 960
Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly
965 970 975
Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile
980 985 990
Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met
995 1000 1005
Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys Thr
1010 1015 1020
Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr Glu
1025 1030 1035 1040
Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050
<210> 3
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Thr Tyr Ile Asp Leu Leu Glu Thr Arg
1 5
Claims (8)
1.一种突变的Cas9蛋白，其特征在于，所述突变的Cas9蛋白源自SEQ ID NO 1；
所述的突变的Cas9蛋白具有剪切基因的活性并且其突变为R338G。
2.一种核酸，其特征在于，所述的核酸是编码权利要求1的Cas9蛋白的核酸。
3.一种CRISPR-Cas9***，其特征在于，使用权利要求1的Cas9蛋白替代野生型Cas9蛋白。
4.权利要求1所述突变的Cas9蛋白的制备方法，其特征在于，按照突变的Cas9蛋白序列人工合成；
或者，在源自Staphylococcus aureus的SaCas9蛋白基础上通过筛选获得，包括以下步骤：
A .通过生物信息学软件辅助筛选SaCas9蛋白中具备免疫原性但不位于其基因剪切功能位点的抗原表位；
B .根据步骤A筛选到的抗原表位构建突变的SaCas9蛋白或合成肽段；
C .使用SaCas9特异抗体阳性的供体血清对突变的SaCas9蛋白进行抗原抗体反应检测并剔除假阳性位点；或者使用SaCas9特异激活T细胞阳性的供体PBMCs检测分析并剔除T细胞激活水平达不到阈值的候选表位；
D .通过基因剪切实验验证突变的SaCas9蛋白，并剔除失去基因剪切作用的SaCas9蛋白。
5.如权利要求4所述的制备方法，其特征在于，
对于B细胞抗原表位，通过生物信息学软件辅助筛选SaCas9蛋白中突变后能够降低免疫原性但不影响其基因剪切功能的抗原表位包括以下步骤：
根据SaCas9蛋白的氨基酸序列和晶体结构模型预测其B细胞抗原表位，使用DISCOTOPE2 .0与IEDB ELLIPRO软件基于连续表位和不连续表位对每个氨基酸残基进行突出指数评分，以PI＝0 .5、残基距离作为阈值，筛选出符合要求的线性表位和构象表位，并获得第一阶段B细胞抗原表位；
对SaCas9蛋白所有氨基酸残基进行亲水性参数、表面可及性参数、抗原性参数和柔韧性参数进行综合预测分析，以亲水性参数≥0，表面可及性参数≥1，抗原性参数≥1作为筛选标准，在所有残基中得到B细胞抗原表位的优势区段，作为第二阶段B细胞抗原表位；
将第一阶段B细胞抗原表位和第二阶段B细胞抗原表位结果进行汇总，选取重合的135个氨基酸位点作为SaCas9蛋白B细胞抗原表位最终的预测结果，即第三阶段B细胞抗原表位；
根据第三阶段B细胞抗原表位，排除功能结构域上的位点后，将其在SaCas9蛋白上分别突变为甘氨酸或丙氨酸，同时构建携带指示标记的切割活性检测***，在该检测***上筛选获得不影响切割活性的突变的SaCas9蛋白，并将相应的17个氨基酸位点作为第四阶段B细胞抗原表位；
获得切割活性不受影响的第四阶段B细胞抗原表位单氨基酸突变的SaCas9蛋白，作为抗原检测其与SaCas9特异抗体阳性的供体血清的抗原抗体反应水平，抗原性下降大于等于50％的B细胞抗原表位作为第五阶段B细胞抗原表位。
6.如权利要求4所述的制备方法，其特征在于，
针对CD8+T细胞抗原表位，通过生物信息学软件辅助筛选SaCas9蛋白中具备细胞免疫原性但不位于基因剪切功能结构域的抗原表位包括以下步骤：
首先，使用IEDB HLA-I软件根据SaCas9蛋白的氨基酸序列预测SaCas9蛋白的CD8+T细胞抗原表位，预测对象分别选择在中国人群中最常见的3种HLA-I型等位基因：HLA-A*11:01(41 .8％)、HLA-A*24:02(31 .8％)和HLA-A*02:01(24 .1％)，针对九肽进行预测，对3种HLA-I分子均选择了结合系数评分前30％的九肽为结合表位肽，选取结合表位肽中重合的50条多肽作为第一阶段CD8+T细胞抗原表位；
其次，使用GalaxyPepDock分子对接模拟软件进行HLA-I分子与50条肽段的对接模拟，分别分析三种HLA-I分子与九肽结合的情况，计算结合自由能，并排除位于基因剪切功能结构域的多肽，得到了预测结果最优的10条多肽作为第二阶段CD8+T细胞抗原表位；
然后，使用SaCas9蛋白特异激活T细胞阳性的供体PBMCs，通过ELISpot方法检测了第二阶段CD8+T细胞抗原表位混合肽对阳性供体PBMCs中T细胞的激活水平，获得能够对T细胞产生激活作用的第三阶段CD8+T细胞抗原表位；
最后，对第三阶段CD8+T细胞抗原表位单肽段分别进行T细胞激活检测，最终得到
SaCas9蛋白适用于中国人群队列的CD8+T细胞优势抗原表位，即第四阶段CD8+T细胞抗原表位。
7.突变的Cas9蛋白的应用，其特征在于，突变的Cas9蛋白在制备基因编辑药物或者试剂中的应用；
所述的突变的Cas9蛋白选自权利要求1的突变的Cas9蛋白。
8.如权利要求7所述的应用，其特征在于，所述的应用包括以下步骤：
(1)将权利要求1的突变的Cas9蛋白的编码核酸与表达载体重组；和/或将权利要求1的突变的Cas9蛋白替代未突变的Cas9蛋白；
(2)与CRISPR-Cas9***其他组成部分共同应用于基因编辑。
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