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使用CRISPR-Cas9系统在胚胎干细胞和普通mar猴的早期胚胎中进行稳健有效的敲入

时间:2019-04-06 08:36:28  来源:南方39助孕

抽象

基因组编辑技术极大地促进了各种细胞和动物的遗传修饰。普通mar猴(Callithrix jacchus),一种表现出高繁殖效率的小型非人灵长类动物,是生物医学研究中广泛使用的动物模型。在普通mar猴中开发基因组编辑技术将进一步增强其效用。在这里,我们报告成功建立了marmoset胚胎干细胞(ESC)的敲入(KI)方法,该方法基于CRISPR-Cas9系统。由同源重组(HR)介导的CRISPR-Cas9的使用增强了mar猴ESC中的KI效率。此外,我们成功地在早期mar猴胚胎中进行了KI。在实验过程中,我们发现mar猴胚胎干细胞中的HR天生就是高效的。这表明mar猴具有DNA双链断裂的修复机制。

介绍

有用于修复DNA双链断裂(DSB的)两个主要的途径1,2,3。一种是非同源末端连接(NHEJ),这是一种容易出错但在哺乳动物细胞中修复DSB的主要途径2。另一种是同源重组(HR),DSB修复的无错误途径,其使用染色单体或外源DNA作为模板3。修复途径的选择取决于物种,细胞类型和细胞周期1

使用HR的基因靶向,即敲入(KI)和敲除(KO),广泛用于疾病建模和基因功能分析。特别地,KI策略用于多种应用,例如在基因组DNA的特定区域引入基因特异性报告基因或错义突变。然而,在哺乳动物细胞中,与由NHEJ 1介导的随机整合相比,HR的频率较低。因此,在这些细胞中,同源重组体更难获得比随机整合克隆4,5

靶向核酸内切酶,例如锌指核酸酶(ZFN),转录激活因子样效应核酸酶(TALEN)和聚集的规则间隔回文重复序列(CRISPR)-Cas9用作NHEJ介导的基因KO 6的基因组编辑工具。此外,由于HR由DSB 7触发,因此靶向核酸内切酶已用于经典基因靶向以提高HR的效率。这种方法非常有效,并且能够通过HR进行遗传修饰,不仅可以用于各种物种的胚胎干细胞(ESC),还可以用于早期胚胎6。在这些技术中,CRISPR-Cas9系统特别方便,因为化脓性链球菌Cas9内切核酸酶仅需要3-bp PAM序列和与靶基因组序列(20个核苷酸)互补的嵌合单指导RNA(gRNA),以在特定基因组基因座8处诱导DSB 。

普通狨猴(狨猴)是作为一个非人类灵长类动物的生物医学模式非常有用,因为它的高育性,短的怀孕期(144天),对人体高生理相似的9,10。我们的团队以前报道的成功产生转基因和KO狨猴的11,12并证明mar猴模型用于研究疾病的效用。但是,生成此类模型的方法仍在开发中,应进行改进以实现更高效的生成。在这里,我们提出了一种HR介导的基因靶向方法,结合了marmoset ESC(cjESCs)中的CRISPR-Cas9基因组编辑技术。使用这种方法,我们在几个不同的cjESCs基因座中获得了高KI效率。此外,我们使用这种方法在早期mar猴胚胎中成功进行KI。令人惊讶的是,我们不仅建立了一种在cjESCs中获得高KI效率的方法,而且我们发现cjESCs具有先天的高HR活性。特别是,在蛋白脂质蛋白1中靶向外显子1的情况下(PLP1)基因,即使不使用CRISPR-Cas9,几乎所有在阳性选择中存活的克隆都是同源重组体。这一独特的特征将有助于mar猴,以及可能的其他非人灵长类动物的疾病建模和基因功能分析的未来研究。

结果

使用CRISPR-Cas9系统评估cjESCs中的KI效率

To test whether the CRISPR-Cas9 system works in marmoset cells, we introduced marmoset-specific gRNA sequences (summarized in Supplementary Table 2) into pSpCas9-2A-Puro (Cas9-gRNA vector; PX459) and evaluated the genomic cleavage activity (GCA) of Cas9 and the gRNAs in cjESCs by transfecting each Cas9-gRNA vector and transiently selecting the transfected cjESCs with puromycin (see Experimental procedures). We confirmed the GCA of Cas9 and all of the gRNAs to be used in the current study (Supplementary Fig. S1a–h).

Next, we decided to target the ACTB gene locus to test whether CRISPR-Cas9 enhances KI efficiency in cjESCs. Previously, by using a promoter-trapping ACTB-EGFP targeting vector (TV) carrying a G418 resistance gene (Fig. 1a)13, we confirmed that most of the G418-resistant and EGFP-positive colonies are homologous recombinants. Therefore, the number of G418-resistant and EGFP-positive colonies are considered to be indexes for KI efficiency.

Figure 1

CRISPR-Cas9增强cjESC中的KI效率。(a)ACTB-EGFP系统的示意图。所述ACTB-EGFP TV窝藏IRES-EGFP-2A-NEO 2.5-kb和5.5-kb的同源臂的狨猴的3'-UTR的周围区域侧翼ACTB基因座上。三个gRNA(ACTB-1,2,3,它们的识别位点显示为剪刀)靶向3'-UTR区域,其不包括在TV中。mar猴ACTB基因的gRNA未检测到电视。黑色细箭头显示用于基因分型PCR的引物结合位点; x,限制酶位点(XbaI); 用于Southern印迹的p,5'-外部探针。(b)G418选择1×10 6转染的ESC 后G418抗性菌落的数目,显示为平均值±sem,n = 3.具有强EGFP荧光(EGFP ++)的菌落数以深灰色显示; 中等EGFP荧光(EGFP +)的菌落数以灰色显示; EGFP阴性菌落(EGFP-)的数量以亮灰色显示。(c)在G418选择后在明视场(BF)或绿色荧光下观察到的EGFP ++(左)和EGFP +(右)集落的代表性图像。比例尺,200微米。(d)EGFP ++和EGFP + cjESC克隆的基因分型PCR分析。M,DNA标记。从相同的凝胶中裁剪单独的图像。(例如)使用5'-外部探针对EGFP ++和EGFP +克隆进行Southern印迹分析。M,DNA标记。从相同的凝胶中裁剪单独的图像。凝胶的整个图像显示在补充图  S14a中。(f)缩短的ACTB-EGFP电视的示意图。(g)选择1×10 6转染的cjESC 后G418抗性菌落的数目,显示为平均值±sem,n = 3.每组用与(b)中相同的颜色表示。* P  <0.05,** P  <0.01。


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为了制备实验,我们首先设计了三种靶向mar 猴ACTB基因的3'-UTR的gRNA(ACTB-1,2和3;补充表2),并  用ACTB-EGFP TV 转染cjESCs ,有或没有每个相应的Cas9-gRNA载体。在用G418阳性选择两周后计数EGFP阳性和阴性集落的数目。在转染含有或不含有每个Cas9-gRNA载体的1×10 6个cjESC后,我们发现在转染Cas9-gRNA的培养物中,G418抗性菌落的数量显着增加(Cas9-gRNA(+))(gRNA1:59.6±14.9) ,gRNA2:90.0±14.4,gRNA3:34.9±12.3;图  1b与未转染的对照培养物(Cas9-gRNA( - ))相比(对照组:8.0±1.2;图  1b)。此外,在Cas9 gRNA(+)组中,我们注意到一些EGFP阳性集落显示出明显更强的EGFP荧光(图1c中的左侧集落  )而不是其它(图1c中的右侧集落  )。因此,我们克隆了六个具有高EGFP荧光(EGFP ++)的EGFP阳性集落和一个具有中等EGFP荧光(EGFP +)的集落。这些克隆的基因分型分析显示,所有EGFP ++克隆都是纯合重组体(图  1d-e),没有任何额外的TV整合(补充图  S2),而EGFP +克隆是杂合重组体(图  1d,e))。这些观察结果表明CRISPR-Cas9基因组编辑增加了cjESCs中的KI效率。

接下来,我们使用三个新构建的具有缩短同源臂的ACTB-EGFP电视来评估KI效率(图  1f)。正如所料,使用缩短的电视导致未用Cas9-gRNA转染的对照组的KI效率降低。然而,当转染Cas9-gRNA(gRNA2)时,我们没有看到KI效率的降低(图  1g)。

另外,为了在不进行阳性选择的情况下估计KI效率,我们还在转染后立即评估转染效率和集落形成效率。用mVenus表达载体(pCXN2-mVenus)转染显示转染效率为32.0±6.3%(n = 5),并且菌落由1.97±0.26%(n = 4)的传代cjESC形成。因此,从1×10 6cjESC,转染约6300个菌落,并预期在阳性选择前形成菌落。因此,在gRNA2和对照组中,转染的集落形成cjESC的靶向效率计算为约1.43%(gRNA2)和0.13%(对照)。为了通过实验验证这种近似计算,我们进行了荧光激活细胞分选(FACS)分析。简而言之,我们用ACTB-EGFP TV和Cas9-gRNA载体(gRNA2)转染cjESCs ,并用嘌呤霉素瞬时选择细胞。进一步扩增这些cjESC,并通过FACS分析EGFP阳性(EGFP(+))细胞的比例。仅使用PX459作为对照。在对照组(gRNA( - ))中,很少有EGFP(+)细胞,计算为约0.18±0.05%(补充图。 S3a)。在gRNA2组(gRNA(+))中,EGFP(+)细胞的百分比增加至1.75±0.17%(补充图  S3b),与对照相比显着增加(P  <0.001;补充图。  S3C)。该结果表明,经历正选择的计数的cjESC集落的数量反映了KI效率,并且使用mVenus表达载体获得的近似计算有助于在一定程度上将计数的集落的数量转化为KI效率。

评估cjESCs中非表达基因的KI效率

We demonstrated the impact of genome editing through targeting of the ACTB gene with a promoter-trap strategy and found that CRISPR-Cas9 indeed increased the number of homologous recombinants. Next, we tested a non-promoter trap strategy at the PLP1 gene locus, which is normally not expressed in cjESCs. PLP1 is a transmembrane proteolipid protein abundantly expressed in oligodendrocytes (OLs)13. Deletion or mutation of the encoding gene causes Pelizaeus-Merzbacher disease (PMD) and spastic paraplegia 214. We constructed four gRNAs targeting different regions which were all in the vicinity of PLP1 exon 1 (PLP1-1, 2, 3, 4; Supplementary Table 2) and a PLP1-EGFP TV, which carries the loxP-flanked PGK-Neo cassette to target the initiation codon of PLP1exon 1 (Fig. 2a). We transfected cjESCs with the PLP1-EGFP TV, with each Cas9-gRNA vector or without (control). When the Cas9-gRNA vectors were used, the numbers of colonies that appeared following the G418 selection increased approximately 8–20 fold (gRNA1: 215.0 ± 119.0, gRNA2: 408.5 ± 128.8, gRNA3: 163.5 ± 12.5, gRNA4: 156.5 ± 22.5, n = 3; Fig. 2b), compared to the control (20.0 ± 9.1, n = 3; Fig. 2b). Because most colonies obtained using the non-promoter trap strategy are usually randomly integrated clones, it was intriguing that the total number of colonies that were homologously recombined were increased when transfecting Cas9-gRNA. We cloned 39 G418-resistant colonies from cjESCs transfected with both the TV and the Cas9-gRNA (gRNA: PLP1-2) vector (Cas9+ clones), and 35 from cjESCs transfected with only TV (Cas9− clones). Genotyping polymerase chain reaction (PCR) revealed that 4/39 (10.3%) Cas9+ clones were homozygous, and 34/39 (87.2%) Cas9+ clones were heterozygous KI clones (Fig. 2c and Supplementary Fig. S4a). Surprisingly, even among the Cas9− clones, 1/35 (2.9%) was homozygous and 31/35 (88.6%) were heterozygous KI clones. This revealed an unexpectedly high KI efficiency with and even without Cas9-gRNA in the G418-resistant cjESC clones. Genotyping PCR was also conducted for the 3′-region of the targeted region; 37/39 (94.9%) Cas9+ clones and 32/35 (91.4%) Cas9− clones were correctly targeted (Supplementary Fig. S4b). In addition, eleven Cas9− clones were subjected to Southern blotting, which confirmed that homologous recombination occurred at the correct locus (Fig. 2d). The genotyping data for G418-resistant clones are summarized in Fig. 2e. Furthermore, to demonstrate the utility of the PLP1-EGFP KI reporter, we differentiated one homozygous-KI cjESC clone (Cas9− #11) into neuronal cells including OLs following excising the loxP-flanked PGK-Neo cassette (Supplementary Fig. S5a–e). Although we failed to detect EGFP fluorescence of the KI cjESCs in an undifferentiated state (Supplementary Fig. S5d, left), EGFP-positive cells emerged from the neurosphere stage (Supplementary Fig. S5d, center). Overall, <5% cells were EGFP-positive among the total differentiated cell on day 70, and at the time point 50–60% of EGFP-positive cells were co-stained with mature OL markers (MBP and GalC; Supplementary Fig. S5f).

Figure 2

评估非活性基因的KI效率。(aPLP1-EGFP构建体的示意图。的PLP1-EGFP TV窝藏3.1-kb和5.0-kb的同源臂,其由围绕所述狨猴的初始密码子的区域的PLP1基因座上。将EGFP编码序列与PLP1基因的5'-UTR的3'末端融合。将聚腺苷酸化信号序列(pA)引入EGFP的末端密码子的下游。另外,将loxP侧翼的PGK-Neo-pA置于EGFP-pA和3'-同源臂之间。针对PLP1附近的gRNA未检测到电视外显子1(PLP-1,2,3,4,显示为剪刀)。细黑箭头表示5'-外部引物和3'-内部引物的位置,用于对5'区域进行基因分型PCR。灰色箭头表示与选择盒同源的5'-引物和用于3'-区域的PCR基因分型的3'-外部引物; b,限制酶位点(Bgl II); p,用于Southern印迹的5'-外部探针。(b)G418选择1×10 6转染的cjESCs 后G418抗性菌落的数量,显示为平均值+ sem,n = 3。(c))G418抗性cjESC克隆的5'-区基因分型PCR。Cas9 +克隆编号以灰色显示(#17-22),Cas9-以黑色显示(#17-22)。剩余克隆的结果显示在补充图  S4a中。何,纯合KI; 他,杂合子KI。(d)使用5'-外部探针对G418抗性Cas9克隆进行Southern印迹分析。(ePLP1外显子1基因靶向数据的总结。* P  <0.05,** P  <0.01。


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为了验证从G418抗性Cas9-cjESC克隆中获得高比率的KI克隆(图  2e),我们还使用从每个孔的G418抗性菌落中提取的大量基因组DNA进行基因分型PCR,所述菌落被转染。PLP1-EGFP TV,有或没有Cas9-gRNA(gRNA:PLP1-2; Cas9(+)或Cas9( - )孔)。结果,在Cas9( - )井(36.9%,34.1%)中,KI带强度归一化为WT带强度,略低于Cas9(+)井(37.2%,48.0%) )(补充图  S6a)。

此外,为了进一步验证采样或分化偏差不影响cjESC的KI效率,我们进行了多能性相关基因OCT4NANOG的定量逆转录-PCR(qRT-PCR)分析。结果显示cjESC在有或没有G418选择的情况下保持相似的未分化状态,这表明高KI比率不是由于在G418选择期间cjESC的分化偏差(补充图  S6b)。另外,在另一个cjESC系中证实了不使用Cas9-gRNA 的PLP1外显子1 的有效KI (补充图  S6c)。这些观察结果表明cjESCs具有先天的高HR活性。

接下来,我们试图引进外显子5,6致病错义突变,和2 PLP1基因15,16。当TV构建时,分别靶向PLP1外显子5或6的PLP1- P216S或PLP1 - SL25T(补充图  S7a)被转染,CRISPR-Cas9增加了G418抗性菌落的数量(补充图  S7b)和KI效率(补充图  S7c,d和S9)。通过多个克隆中的DNA测序证实了正确基因座中的精确KI,但发现一些克隆在KI等位基因中具有镶嵌或WT序列(补充图  S8a,b)。和表  5)。同样,在将PLP1- A39T构建体转染到PLP1外显子2(补充图  S7e)的情况下,嘌呤霉素抗性菌落的数量(补充图  S7f)和KI效率增加(补充图  S7g和S9)。转染了gRNA2和gRNA3(分别为PLP1-CDS2-2和PLP1-CDS2-3)的KI克隆中没有异常序列,而在转染了gRNA1和gRNA4(PLP1-CDS2-1和PLP1)的克隆中发现了一些镶嵌现象。 -CDS2-4,分别)克隆(补充图  S8c和补充表  5)。这些结果总结在补充图中。 S7h。

FOXP2基因的人源化

由于PLP1是X染色体基因,我们接下来在FOXP2(一种常染色体基因)中测试了我们的方法。叉头框蛋白P2(FOXP2)基因是与肺和皮质发育的转录因子17,18。此外,在基因数进化和致病性突变与人类的语言能力有关19,20

我们为FOXP2内含子8 构建了3个gRNA,为外显子8 构建了1个gRNA (分别为FOXP2-1,2,3,4;补充表  2)和FOXP2 TV,其中包含两个人特异性氨基酸序列(T301N和N323S)20FOXP2外显子8和PGK-PuroTK-pA盒中,侧翼为FLP识别靶位点(FRT)21(图  3a)。我们用TV转染cjESCs,有或没有每个Cas9-gRNA载体。CRISPR-Cas9的使用使嘌呤霉素抗性菌落的数量增加了55-191倍(图  3b)。此外,0/6(0%)的Cas9克隆,6/6(100%)的Cas9 + gRNA1,6 / 6(100%)的Cas9 + gRNA2,6 / 6(100%)的Cas9 + gRNA3和28 / 33个(84.8%)Cas9 + gRNA4克隆是KI克隆(图  3c)。另外,用Cas9和gRNA4转染导致纯合KI克隆的有效产生(8 / 33,24.2%;图  3c和补充图  S10)。通过Southern印迹也对8个克隆进行基因分型。得到的数据与基因分型PCR的数据相匹配,除了发生异常重组的一个克隆(gRNA4#19)(图  3d))。然而,当使用靶向内含子8(FOXP2-1,2,3)的Cas9和gRNA时,来自PCR分析(7.0kb)的KI等位基因条带的DNA测序分析显示KI等位基因中的外显子8的序列是WT或马赛克。 (混合的WT和人源化序列;图  3e,底部和补充表  5)。另一方面,通过用TV和靶向外显子8的Cas9-gRNA转染cjESC而产生的KI克隆(FOXP2-4)在所有KI等位基因的外显子8中具有人源化序列(图  3e,顶部和补充表  5)。 。嘌呤霉素抗性克隆的基因分型数据显示在图  3f中。

图3

Humanization of the FOXP2 gene. (a) Schematic diagram of FOXP2 targeting. The FOXP2 TV harboured FRT-flanked PGK-PuroTK-pA cassette between the 2.0-kb and 1.3-kb homology arms for intron 8 of the FOXP2 gene locus. The magenta and orange arrowheads indicate two human-specific substitutions, T301N and N323S, respectively; the sequences were introduced to the FOXP2 exon 8 locus of the TV. The TV was not detected by gRNAs for the marmoset FOXP2 gene (FOXP2-1, 2, 3, 4, shown as scissors). Thin black arrows indicate the 5′- and 3′-external primers used for genotyping PCR; v, a restriction enzyme site (EcoRV); p, the 5′-external probe for Southern blotting. (b) The number of puromycin-resistant colonies following selection of 1 × 106 transfected cjESCs, shown as the mean + s.e.m., n = 3. (c) Genotyping PCR analysis of the puromycin-resistant ESC clones (FOXP2 humanization). Among the clones of gRNA4, six representative clones (#11–15) are shown, and the remaining clones are shown in Supplementary Fig. S10. (d) Southern blotting analysis of puromycin-resistant cjESC clones using the 5′-external probe. The data of eight clones (gRNA1 #1, gRNA2 #1, gRNA3 #1, control #1, gRNA4 #12, 8, 15, 28) genotyped by Southern blotting matched that of genotyping PCR, except for one aberrantly recombinated clone (gRNA4 #19). (e) DNA sequencing analysis of the KI alleles. A representative mutated KI clone (gRNA4 #12) and a mosaic clone (gRNA2 #4) are shown. (f) Summary of the FOXP2 gene targeting data. *P < 0.05, **P < 0.01.


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In conclusion, by using the FOXP2 TV and Cas9-gRNA (gRNA: FOXP2-4) vector, we were able to successfully generate homozygous KI clones harbouring human-specific mutations at the correct site.

Evaluation of KI efficiency in early-stage marmoset embryo

We have demonstrated that the developed KI method for cjESCs resulted in high KI efficiency. Next, we attempted to apply this KI method to early-stage marmoset embryos (Fig. 4a). For this experiment, we selected the PLP1 exon 2 KI construct (Fig. 4b), since this construct enabled homologous recombination in cjESCs the most efficiently (Supplementary Figs S7e–h, S8c, S9, and Table 5) among the experiments described above. In addition, the PLP1-CDS2-2 gRNA induced KI the most efficiently among the four gRNAs targeting PLP1exon 2 (Supplementary Fig. S7f) with the least concern of mosaicism in cjESCs (Supplementary Table 5), so we used the gRNA for further experiments with early-stage embryos. We used the PLP1-P15L TV encoding the pathogenic P15L substitution22 for PLP1 exon 2 and harbouring a SacI restriction enzyme site instead of an ApaI restriction enzyme site in the WT allele (Fig. 4b). We performed genotyping PCR using the entire genomic DNA extracted from each embryo which developed to the 8-cell stage or beyond (Fig. 4a, right). The PCR products were subjected to restriction fragment-length polymorphism (RFLP) analysis and DNA sequencing after subcloning the product into a blunt vector. The RFLP analysis of the KI using the TV was first validated in cjESCs (Fig. S11a and b).

Figure 4

Evaluation of KI efficiency in the early-stage marmoset embryo. (a) Schematic of the gene targeting experiment using early-stage marmoset embryos. 2PN, the two-pronuclear stage. Scale bar, 50 μm. (b) Schematic of the PLP1-P15L KI construct in the embryos. Grey arrowhead indicates the location of sequence resulting in the P15L substitution and the SacI site in the TV. Scissors indicate the DSB site. Thin blue arrow indicates the gRNA-targeted sequence (including the PAM sequence of streptococcus pyogenes Cas9; NGG). Black arrowhead indicates the Cas9-cleaved sequence site. (c) RFLP analysis of the microinjected embryos (#12 and #14–17, the remaining results are shown in Supplementary Fig. S12b). White arrowheads indicate detection of the KI allele by SacI digestion of the PCR fragments. (d) DNA sequence analysis of the KI allele of embryos #12 and #14. Results for the remaining embryos are appended in Supplementary Fig. S12c.


Full size image

Based on preliminary experiments (data not shown), we optimized the concentration of each component in the microinjection solution. The optimized solution consisted of the Cas9 protein (100 ng/μl), annealed crRNA and tracrRNA (50 ng/μl), and TV (100 ng/μl) in nuclease-free water, which were injected into the cytoplasm of two pronuclear-stage (2PN) zygotes (Fig. 4a, left). For the microinjection, 38 embryos were used; 18/38 (48.6%) embryos developed to the 8-cell stage or beyond; and 17 of them were genotyped. Surprisingly, under this condition, all the genotyped embryos were genetically modified (Fig. S12a and c). In 4/17 (24%) embryos (#2, #9, #12, #14), the PCR fragment was digested by SacI, which confirmed KI (Fig. 4c, Supplementary S12b). KI was further validated with DNA sequencing of the subcloned PCR fragments (Fig. 4d and Supplementary Fig. S12c). At least 7 subclones per embryo were analyzed. However, more than two sequence variations were found in 7/17 (41%) embryos, which showed that mosaicism occurred in these embryos (Supplementary Fig S12c). These results are summarized in Supplementary Fig. S12a.

Furthermore, we utilized a zygote-electroporation method for early-stage marmoset embryos. Referring to previous studies performed in mice embryos23,24, we set an electroporation condition for marmoset embryos. Components of the electroporation solution included the Cas9 protein (100 ng/μl), annealed crRNA and tracrRNA (50 ng/μl), and the TV (100 ng/μl) in 1x OPTI-MEM. Additionally, since successful KI using a double-stranded DNA vector has never been achieved by electroporation in rodent embryos23,24,25, we utilized a 200-bp single-stranded oligonucleotide (ssODN) which overlaps the DSB site of the PLP1-CDS2-2 gRNA, and encodes the P15L substitution, SacI site, and silent mutations, included for identifying the type of KI template transfected in each embryo. The ssODN-mediated KI was validated in cjESCs by RFLP analysis. The ssODN showed lower KI efficiency (2.8% and 4.1%) compared to that with the TV (3.9% and 8.3%) in our condition (n = 2; Supplementary Fig. S11b), different from a previous report showing an increased efficiency in KI for ssODN relative to TV26but consistent with a previous study using murine ESCs27, However, further experiments would be required to make a definitive conclusion on this subject. The sequence of the ssODN is appended in the Supplementary Information. For these experiments, 7 embryos were used, 6 (86%) of them developed to the 8-cell stage or beyond, and subsequently genotyped. We found that 4/6 (66%) embryos carried genetically modified alleles, and 2/6 (33%) embryos had KI alleles resulting from transfection of either the ssODN or the TV, which were detected by DNA sequencing and RFLP analysis (Supplementary Fig. S13b and c). The data are summarized in Supplementary Fig. S13a.

Thus, by using the microinjection and electroporation conditions we developed and optimized, we succeeded in efficiently introducing gene modifications in marmoset embryos, resulting in either KI or KO.

Discussion

In the current study, we established a gene targeting method for cjESCs and early-stage embryos, using CRISPR-Cas9 for directed DSB. This method dramatically increased the number of colonies that survived positive selection, and enabled bi-allelic homologous recombination. Furthermore, this method is robust, since KI of several genetic loci, including genes that are normally not expressed in undifferentiated cjESCs, was obtained with efficiencies similar or even higher than that of ACTB. Although the KI efficiency among total transfected cjESCs were low, experimentally shown to be between 1.8–8.3% with Cas9-gRNA (Supplementary Figs S3b and S11b), the percentage of KI clones among cjESC clones that survived positive selection was over 80% in most cases (Figs 2e, 3f and Supplementary Fig. S7h), which is higher than that of previous studies recently reported using a similar strategy in human ESCs and induced pluripotent stem cells (iPSCs)28,29,30,31,32,33and in macaque monkey ESCs (less than 50%)34. Additionally, in our method, we succeeded in obtaining over 10% of homozygous-KI clones in most cases (Figs 2e, 3f and Supplementary Fig. S7h). This was surprising, as homozygous KIs has been considered to be difficult to obtain using the conventional KI system in human iPSCs33.

We also observed an innate high HR activity of cjESCs, which may have also contributed to the extremely high KI efficiency revealed in this study. In the course of the current study, we observed a high KI ratio when targeting PLP1 exon 1, even in the absence of site-directed DSB with Cas9-gRNA. We were able to obtain KI clones with 88.6% (31/35 clones) efficiency without negative selection, one of which was a homozygous KI clone (Fig. 2e and Supplementary Fig. S4). Since gene targeting efficiency of PLP1 exon 1 in mouse ESCs was 2% using a similar construct13, we consider our result in cjESCs to be extraordinary. Although this high efficiency was not observed at other loci without Cas9-gRNA, an HR bias may have occurred in cjESCs. Further studies to explore the mechanism of HR in cjESCs will be useful for improving the KI efficiency in other species.

由于PLP1基因对OLs 35形成的髓磷脂的稳定性至关重要,因此其突变,缺失或复制导致中枢神经系统14的功能损害。一些PLP1错义突变导致严重的表型,但该基因的无效突变或遗传缺失导致轻度表型36。此外,PLP1基因被认为与在小鼠精神分裂症和相关联的人37,38。因此,能够在PLP1中实现诸如KO的遗传修饰或引入错义突变在当前研究中进行的非人灵长类动物基因将有助于产生用于分析运动功能和更高脑功能的新疾病模型,其可用于在临床前环境中测试候选药物和细胞植入。

FOXP2最初被确定为遗传性语言障碍的基因19。因此,该基因可能与进化过程中人类的语言习得有关,因为两个氨基酸残基(N303和S325)对人FOXP2具有特异性。人源化FOXP2小鼠表现出和学习纹状体神经解剖学的变化增强的存储器39,40。由于一些组建议在非人类灵长类,包括狨猴中在Broca区的同系物的存在41,42,43FOXP2将是在狨基因修饰一个有趣的候选者。

此外,我们还在普通mar猴的早期胚胎中建立了KI技术,其基于显微注射和电穿孔方法。我们成功获得了约30%效率的KI胚胎。将来,这项技术可用于生成KI mar猴。

总之,我们为mar猴胚胎干细胞和早期胚胎开发了一种有效的KI方法。该方法可用于产生KI动物并用于使用非人灵长类动物模型体外分析基因功能。此外,应该彻底研究在cjESC中DSB之后发生并且在本文提出的几个实验中观察到的HR偏差,以用于KI技术的未来发展。当前研究中的发现将有助于使用非人类灵长类物种(mar猴)作为桥接模型来填补小鼠和人类之间的空白。

实验步骤

动物

所有动物实验方案均按照美国国立卫生研究院和日本教育,文化,体育,科学和技术部(MEXT)制定的实验动物指南进行,并获得Institutional Animal批准。 RIKEN的护理和使用委员会(批准号H27-2-306(4))。动物护理按照国家研究委员会(NRC)实验动物护理和使用指南(2011)进行。本研究中使用的Mar猴是2-6岁(平均体重250至450克)。mar猴在温暖潮湿的条件下(25°C,65%)配对/家庭饲养。总共有30只雌性mar猴用作卵母细胞供体,11只雄性mar猴用作精子供体。mar猴是从RIKEN研究所的内部繁殖地获得的。将卵母细胞供体与血管扩张的雄性成对保持。卵母细胞和精子的收集,和体外授精(IVF)如先前所述进行12。简言之,对于卵母细胞采集,监测血浆孕酮水平的卵母细胞供体肌内注射重组人卵泡刺激素(FSH,25 IU; Fuji Pharma)9天,然后肌内注射人绒毛膜促性腺激素(hCG)在第10天,75 IU; ASKA Pharmaceutical)在第11天(hCG注射后16-20小时),激素处理的雌性mar猴用0.04mg / kg美托咪定(Nippon Zenyaku)预先麻醉,0.40mg / kg布托啡诺(Meiji Seika Pharma)和0.40 mg / kg咪达唑仑(Astellas Pharma)。从麻醉的动物手术收集卵母细胞。在手术期间,用异氟烷(Sumitomo Dainippon Pharma)将mar猴吸入麻醉。将收集的卵母细胞在猪卵母细胞培养基(功能性肽研究所)中于38℃温育。对于IVF,从健康的雄性mar猴收集精子。对于授精,将每个卵母细胞与3.6×10孵育在一滴TYH培养基(LSI Medience)中4个精子在38℃下保持16小时。

细胞培养

两种常见的mar猴ES细胞系,40号(雌性,46XX)和DSY127(雄性,46XY)用于本研究。40号线先前成立44号,DSY127由Sumitomo Dainippon Pharma Co.,Ltd。(日本东京)友情提供。如前所述培养这些细胞45。简而言之,cjESCs在30 Gy照射的小鼠胚胎成纤维细胞(MEF)中培养在ES培养基(ESM)中,该培养基由1x KnockOut DMEM(Thermo Fisher)组成,补充有20%KnockOut血清替代物(Thermo Fisher),1mM L-谷氨酰胺(Nakalai) Tesque),1%非必需氨基酸(Sigma),0.2mM 2-巯基乙醇(Sigma)和10ng / ml成纤维细胞生长因子2(Peprotech)。本研究中产生的KI cjESC品系将由相应的作者根据要求分发。

转染和基因分型

用于cjESC中的KI实验的双链DNA靶向载体(TV)在转染前通过一次切割限制酶线性化。在Tris-HCl-EDTA缓冲液(pH8.0)中以1μg/μl制备每种Cas9-gRNA载体和线性化TV。对于转染,转染总共10μg的DNA,其由TV和Cas9-gRNA载体以4:1的摩尔比组成。对于cjESC转染,将DNA载体(总共10μg),lipofectamine-LTX PLUS试剂(2.5μl; Thermo Fisher)和LTX试剂(25μl; Thermo Fisher)稀释于500μlOPTI-MEM(Thermo Fisher)中,并加入在6孔板的一个孔中培养的亚融合的cjESC中。转染后24小时,将细胞解离并计数。1×10 6将cjESC悬浮于含有Y-27632(10μM; Merck Millipore)的ESM中,并重新接种到对G418或嘌呤霉素具有抗性的新饲养细胞上(第1天)。在第3天,将培养基更换为含有G418(50μg/ ml; Thermo Fisher)或嘌呤霉素(1μg/ ml; Thermo Fisher)和Y-27632(10μM)的ESM。从第5天开始从培养基中除去Y-27632。2周后,计数抗药性菌落并挑选进一步克隆。

For genotyping by PCR and Southern blotting, the cloned ESCs were lysed overnight at 55 °C in cell lysis buffer consisting of Tris-HCl (0.2 M), EDTA (10 mM), SDS (0.2%) and NaCl (0.2 M) in nuclease-free water with proteinase K (10 μg/ml). Genomic DNA was purified using a standard method with phenol-chloroform and ethanol. PrimeSTAR Max DNA polymerase (Takara) was used for genotyping PCR, according to the manufacturer’s instructions. PCR was performed as follows: 30 s at 94 °C; 35 cycles of 10 s at 98 °C and 8 min at 68 °C; then 10 min at 68 °C; and a final incubation at 4 °C until gel electrophoresis. The primers are listed in Supplementary Table 1. Southern blotting was performed as described previously45. For digestion of genomic DNA, we used XbaI (ACTB-EGFP), BglII (PLP1-EGFP) and EcoRV (FOXP2PLP1-P216S, S253T and A39T) (purchased from Takara or NEB). The entire images of the gels in Southern blotting analysis were appended in Supplementary Fig. S14.

Transient selection

The procedure for the transfection of cjESCs is described above. After transfecting each TV or ssODN (8 μg) with the Cas9-gRNA vector (2 μg), the cjESCs were re-seeded onto new feeder cells (day 1). On day 2, the medium was changed to ESM. On day 3, the medium was changed to ESM containing puromycin (0.2 μg/ml) and Y-27632 (10 μM). On day 5, Y-27632 was removed from the medium. From day 6, the medium was changed to ESM every other day for further expansion.

FACS analysis

FACS analysis was performed using FACSVerse flow cytometer (BD) with FACSuite software (BD) according to the manufacturer’s instructions. In brief, the cjESCs were expanded following transient selection, and the cells were suspended in FACS buffer consisting of fetal bovine serum (1%), EDTA (5 mM) and Y-27632 (10 μM) in 1x PBS buffer. 0.1% Propidium iodide staining solution (PI; Sigma) was used to remove dead cells.

Genomic cleavage assay

Cas9-guided genomic cleavage assay was performed using GeneArt genomic cleavage detection kit (Thermo Fisher) according to the manufacturer’s instructions. Briefly, each Cas9-gRNA vector (10 μg) was introduced into cjESCs and then the cells were transiently selected. Genomic DNA was extracted from the cells for PCR. The resulting PCR mixtures were re-annealed under the following conditions: 5 min at 95 °C; 85 °C to 25 °C at −2 °C/s ramp speed; and 25 °C to 4 °C at −0.1 °C/s ramp speed. The re-annealed solutions were directly used for the cleavage reaction.

Vector construction

Cas9-gRNA vectors

CRISPR-direct46 was used to design gRNA for marmoset genomic DNA. The gRNA sequences are listed in Supplementary Table 2. For the Cas9-gRNA vector, pSpCas9(BB)-2A-Puro (PX459) was used (a gift from Feng Zhang; Addgene plasmid # 48139).

TVs

The ACTB-EGFP vector was constructed previously45. The shortened ACTB-EGFP vectors were constructed based on the ACTB-EGFP vector. The genomic sequences of the putative marmoset PLP1 and FOXP2genes were obtained from the C. jacchus genome database (Callithrix_jacchus-2.0.2: The Genome Sequencing Center at Washington University School of Medicine in St. Louis, https://genome.wustl.edu/pub/organism/Primate/Callithrix_jacchus/). To construct the PLP1-EGFP vector, a 3.2-kb fragment containing a region spanning upstream of the PLP1 gene to the PLP1 initiation codon, and a 5.0-kb fragment containing PLP1 intron 1 were ligated with the vector pHNEO-EGFP, which harbours promoter-less EGFP, bovine growth hormone polyadenylation signal (pA), and floxed G418-resistance gene (Neo) under the mouse Pgk-1 promoter (PGK).

To construct the PLP1-P216S and PLP1-S253T vectors, a 2.7-kb fragment containing PLP1 introns 2 to 5, and a 4.2-kb fragment containing PLP1intron 5 and the downstream region of the PLP1 gene, were ligated with pSINTK. The 2.7-kb fragment was mutagenized by PCR using specific primers to obtain the sequence encoding the Pro216Ser substitution (CCT > TCT), or the 4.2-kb fragment was mutagenized to obtain the sequence encoding the Ser253Thr substitution (TCC > ACC).

To construct the PLP1-A39T and PLP1-P15L vectors, a 2-kb fragment containing a region from PLP1 intron 1 to intron 2, and a 1.4-kb fragment spanning PLP1 intron 2 to intron 4, were subcloned into pDONR vectors, and introduced into pDEST-R3R4(R) with pENTR-L1-PGK-PuroTK-pA-L247. The 2-kb fragment was mutagenized by PCR to generate A39T or P15L substitutions, and silent mutations to render the vectors to become undetectable by the gRNAs. For gene targeting experiments in the embryos, the PGK-PuroTK cassette was removed from the PLP1-P15L vector by Cre recombinase (NEB).

To construct the FOXP2 TV, 2.6-kb and 1.3-kb fragments were subcloned into pDONR vectors, and introduced into pDEST-R3R4(R) with pENTR-L1-PGK-PuroTK-pA-L247. The 2.6-kb fragment was mutagenized to encode human-specific variants (Thr301Asn and Asn323Ser).

All vectors were purified using plasmid DNA purification kit (Qiagen). The vectors used in the current study are listed in Supplementary Table 3. The listed vectors will be provided by Addgene (https://www.addgene.org) or the corresponding authors upon request.

Neuronal induction

The cjESCs were induced into neuronal cells, including OLs, using a previously described method with slight modifications48. Briefly, dorsomorphin (3 μM; Sigma), SB431542 (3 μM; Tocris Bioscience) and CHIR99021 (3 μM; Wako) were added at days 1–3 of embryoid body formation. Immunochemical analysis was performed using Hoechst 33258 (Sigma) and the antibodies listed in Supplementary Table 4. The detailed experimental protocols will be provided by the corresponding authors upon request.

Microinjection of the embryos

Marmoset embryos at the 2PN stage were prepared as described previously12,49. The microinjection solution consisted of annealed crRNA and tracrRNA (50 ng/μl; IDT), Cas9 protein (100 ng/μl; IDT) and TV (PLP1-P15L; 100 ng/μl), which were suspended in nuclease-free water. The TV was purified using QIAquick PCR purification kit (Qiagen). Approximately 5–10 pl of the injection solution was injected into the cytoplasm of the pronuclear stage embryos in M2 medium (Sigma). Following the microinjection, the embryos were cultured in ORIGIO sequential cleavage medium (Origio). The embryos that developed normally past the 8-cell stage were used for genotyping. The KAPA mouse genotyping kit (Kapa Biosystems) and PrimeSTAR Max DNA polymerase were used for embryo genotyping. Briefly, each developed embryo was washed in PBS drop once and then transferred into the extraction solution (3 μl) consisting of extraction buffer (1×) and extraction enzyme (2%), and was placed in the following conditions: 75 °C for 10 min, 95 °C for 5 min, and a final incubation at 4 °C. After extraction, PCR solution (22 μl) was added to the extract solution and centrifuged briefly before PCR reaction. The final PCR mixture (25 μl) contained the PrimeSTAR Max premix (1×) and primers (1.6 μM each). PCR was performed by temperature cycling as follows: 30 s at 94 °C; 40 cycle of 10 s at 98 °C and 150 s at 68 °C; 10 min at 68 °C; and a final incubation at 4 °C. A portion (4 μl) of the PCR solution was used for electrophoresis on 1% agarose gel, and the 1.5–2.2-kb DNA product band was extracted from the gel. The extracted PCR fragments were purified using a phenol-chloroform and ethanol method, and subcloned into pCR-BluntII-TOPO (Thermo Fisher) utilizing DH5αcompetent cells (Takara). Each cloned vector was sequenced using the BigDye Terminator v1.1 Cycle sequencing kit (Thermo Fisher) with the 3130xl DNA Analyzer (Applied Biosystems). For the RFLP analysis, a portion (4 μl) of the PCR mixture was used. The RFLP solution (20 μl) contained buffer L (1×), and ApaI or SacI (1 μl; Takara). The RFLP reaction was performed as follows: 4 hours at 37 °C, followed by 20 min at 80 °C, and a final incubation at 4 °C until gel electrophoresis.

Electroporation of the embryos

NEPA21超级电穿孔仪和CUY505P5电极(Nepa Gene)用于电穿孔。将2PN阶段的Mar猴胚胎移植到含有Cas9蛋白(100ng /μl),退火的crRNA和tracrRNA(总共25ng /μl),TV(100ng /μl)和ssODN(100ng / 100)的电穿孔溶液中。 μ1)在OPTI-MEM(1×; Thermo Fisher)中。电穿孔条件如下。打孔脉冲:225 V,2脉冲宽度,50 ms脉冲间隔,4个脉冲,10%衰减率,+。传输脉冲:20 V,50 ms脉冲宽度,50 ms脉冲间隔,5个脉冲,40%衰减率,+ / - 。将电阻值调整为约。电穿孔前500Ω。电穿孔后,将胚在ORIG10序列切割培养基中培养直至发育超过8细胞阶段。

RT-PCR和qRT-PCR

根据制造商的方案使用RNeasy mini试剂盒(Qiagen)分离RNA。在ReverTra Ace qPCR RT主混合物(Toyobo)中逆转录总RNA(1.0μg)。将得到的cDNA在无核酸酶的水中稀释(至4ng /μl)。根据制造商的说明书使用PrimeSTAR Max DNA聚合酶进行RT-PCR。根据制造商的说明书,使用Viia 7(Applied Biosystems)上的TB Green Premix Ex Taq II(Takara)进行qRT-PCR。使用的引物列于补充表  1中。

DNA测序

使用BigDye Terminator v1.1循环测序试剂盒(Thermo Fisher)和3130xl遗传分析仪(Applied Biosystems)进行DNA测序分析。使用Snap Gene软件(GSL Biotech)说明图中呈现的序列数据。

统计分析

所有数据均表示为平均值±sem使用Student's t检验比较平均值之间的差异。差异被认为是显着的,P  <0.05。

附加信息

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