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智科眼: 双语看懂新科研
In(In) a(a) study(研究) published(发表) in(in) Nature(自然) on(on) June(6月) 10,(10,) researchers(研究人员) from(from) Dr(博士) .(.) Yang() Hui(Hui)'s('s) Lab(实验室) at(at) the Institute(研究所) of(of) Neuroscience(神经科学) of(of) the Chinese(中国) Academy(科学院) of(of) Sciences(科学) (CAS),((CAS),) and() collaborators(合作者) from(from) the CAS-MPG(CAS-MPG) Partner(伙伴) Institute(研究所) for(for) Computational(计算) Biology(生物学) of(of) CAS(CAS) and() Sichuan(四川) University(大学) demonstrated(证明了) that(that) DNA(DNA) base(碱基) editors(编辑器) generated(产生) tens() of(of) thousands() of(of) off(off)-()target() RNA(RNA) single() nucleotide(核苷酸) variants(变体) (SNVs)((SNVs)) and() these(这些) off(off)-()target() SNVs(SNVs) could(可以) be(be) eliminated(消除) by(by) introducing(引入) point mutations(点突变) to(to) the deaminases(脱氨酶).(.)
This() study(研究) revealed(揭示) a(a) previously(先前) overlooked(被忽视) aspect(方面) of(of) the risk(风险) of(of) DNA(DNA) base(基础) editors(编辑) and() further(进一步) provided(提供了) a(a) solution(解决) to(to) the problem(问题) by(by) engineering(工程) deaminases(脱氨酶).(.)
DNA(DNA) base(碱基)-()editing(编辑) methods(方法) have(have) enabled() direct(直接) point mutation(点突变) correction(校正) in(in) genomic DNA(基因组DNA) without(没有) generating(产生) any(任何) double-strand breaks(双链断裂) (DSBs),((DSBs),) but() the potential(潜在) off(off)-()target() effects(效应) have(have) limited(限制) the application(应用) of(of) these(这些) methods(方法).(.) Adeno(Adeno)-()associated(相关) viruses(病毒) (AAV)((AAV)) are(are) the most() common(常用) delivery system(输送系统) for(for) DNA(DNA) editing(编辑) gene therapies(基因治疗).(.) Since(由于) these(这些) viruses(病毒) can() sustain(维持) long-term(长期) gene expression(基因表达) in vivo(体内),(,) the extent(程度) of(of) potential(潜在) RNA(RNA) off(off)-()target() effects(效应) induced(诱导) by(by) DNA(DNA) base(碱基) editors(编辑) is(is) of(of) great(非常) concern(关注) for(for) their() clinical application(临床应用).(.)
Several() previous(以前) studies(研究) have(have) evaluated(评估) off(off)-()target() mutations(突变) in(in) genomic DNA(基因组DNA) by(by) DNA(DNA) base(碱基) editors(编辑).(.) Meanwhile(同时),(,) the deaminases(脱氨酶) integral(积分) to(to) commonly(通常) used(使用) DNA(DNA) base(碱基) editors(编辑器) often() exhibit(展示) RNA(RNA) binding(结合) activities(活性).(.) For example(例如),(,) the cytosine(胞嘧啶) deaminase(脱氨酶) APOBEC1(APOBEC1) used(使用) in(in) cytosine(胞嘧啶) base(碱基) editors(编辑器) (CBEs)((CBEs)) was(was) found(发现) to(to) target(靶向) both(both) DNA(DNA) and(并且) RNA,(RNA,) and() the adenine deaminase(腺嘌呤脱氨酶) TadA(TadA) used(使用) in(in) adenine(腺嘌呤) base(碱基) editors(编辑器) (ABEs)((ABEs)) was(was) found(发现) to(to) induce(诱导) site(位点)-()specific(特异性) inosine(肌苷) formation(形成) on(on) RNA.(RNA.) However(然而),(,) any(任何) potential(潜在) RNA(RNA) mutations(突变) caused(引起) by(by) DNA(DNA) base(碱基) editors(编辑) had(had) not(尚未) been(been) evaluated(评估).(.)
In order to(为了) evaluate(探讨) the off(off)-()target() effect(效应) of(of) DNA(DNA) base(碱基) editors(编辑器) at(at) the level(水平) of(of) RNA,(RNA,) the researchers(研究人员) counted(计算) the off(off)-()target() RNA(RNA) SNVs(SNVs) in(in) each(每个) replicate(复制) of(of) CBE-(CBE-) or() ABE-(ABE-)treated(处理) cells(细胞),(,) and() then(然后) explored(探索) the possibility(可能性) of(of) eliminating(消除) the off(off)-()target() RNA(RNA) SNVs(SNVs) by(by) engineering(工程) deaminases(脱氨酶) of(of) DNA(DNA) base(碱基) editors(编辑).(.)
They(他们) transfected(转染) one(一种) type(类型) of(of) CBE,(CBE,) BE3(BE3) (APOBEC1-nCas9-UGI),((APOBEC1-nCas9-UGI),) or() ABE,(ABE,) ABE7.10(ABE7.10) (TadA-TadA*-nCas9),((TadA-TadA*-nCas9),) together(一起) with(with) GFP(GFP) and() with(with) or() without(没有) single()-()guide(指导) RNA(RNA) (sgRNA),((sgRNA),) into(into) HEK293T-(HEK293T-)cultured cells(培养细胞).(.) After(After) validating(验证) the high() on-(on-)target() efficiency(效率) of(of) DNA(DNA) editing(编辑) by(by) both(both) BE3(BE3) and() ABE7.10(ABE7.10) in(in) these(这些) HEK293T(HEK293T) cells(细胞),(,) they(他们) performed(进行) RNA-(RNA-)seq(seq) at(at) an(一个) average(平均) depth(深度) of(of) 125X(125X) on(on) these(这些) samples(样品) and() quantitively(定量) evaluated(评估) the RNA(RNA) SNVs(SNVs) in(in) each(每个) replicate(复制).(.)
The on-(on-)target() editing(编辑) efficiency(效率) was(was) evaluated(评估) in(in) each(每个) replicate(复制) of(of) the CBE-(CBE-) or() ABE-(ABE-)treated(处理) cells(细胞) to(to) guarantee(保证) efficient(有效) editing(编辑).(.) Then(然后) the number(数量) of(of) off(off)-()target() RNA(RNA) SNVs(SNVs) in(in) CBE-(CBE-) and() ABE-(ABE-)treated(处理) groups() was(was) compared(比较) with(with) the GFP-(GFP-)only() control group(对照组).(.) They(他们) found(发现) strikingly(引人注目) higher(更高) numbers(数量) of(of) RNA(RNA) SNVs(SNVs) in(in) DNA(DNA) base(碱基) editor(编辑)-()treated(处理) cells(细胞).(.)
Furthermore(此外),(,) the researchers(研究人员) found(发现) that(that) the mutation(突变) bias(偏差) in(in) BE3-(BE3-) or() ABE7.10-(ABE7.10-)treated(处理) cells(细胞) was(was) the same(相同) as(as) that(that) of(of) APOBEC1(APOBEC1) or() TadA,(TadA,) respectively(分别),(,) indicating(表明) the off(off)-()target() effects(效应) were(were) caused(引起) by(by) the overexpression(过表达) of(of) DNA(DNA) base(碱基) editors(编辑).(.) They(他们) also() identified(鉴定) CBE-(CBE-) and() ABE-(ABE-)specific(特异性) motifs(基序) and() genetic(遗传) regions(区域) of(of) these(这些) off(off)-()target() RNA(RNA) SNVs.(SNVs.)
To(To) eliminate(消除) the RNA(RNA) off(off)-()target() activity(活性) of(of) base(基础) editors(编辑),(,) they(他们) examined(检查) the effect(效果) of(of) introducing(引入) point mutations(点突变) on(on) APOBEC1(APOBEC1) or() TadA(TadA) .(.) Three(三种) high-fidelity(高真实性) variants(变体),(,) BE3W90Y+R126E,(BE3W90Y+R126E,) BE3(BE3) (hA3AR128A)((hA3AR128A)) and() BE3(BE3) (hA3AY130F),((hA3AY130F),) reduced(降低) RNA(RNA) off(off)-()target() SNVs(SNVs) to(to) the base level(基准面).(.) Similarly(类似),(,) an(一个) ABE(ABE) variant(变体) ABE7.10F148A(ABE7.10F148A) also() showed(显示) complete(完全) elimination(消除) of(of) off(off)-()target() effects(效应).(.)
This() study(研究) obtained(获得) both(both) high-fidelity(高真实性) variants(变体) for(for) both(both) CBEs(这些CBE) and() ABEs(ABEs) by(by) introducing(引入) point mutations(点突变) to(to) the deaminases(脱氨酶) and() provided(提供了) a(a) proposed(提出) method(方法) using(使用) rational(合理) engineering(工程) to(to) increase(提高) the specificity(特异性) of(of) base(基础) editors(编辑).(.)

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