God does not play dice, and neither does CRISPR/Cas9.
God does not play dice, and neither does CRISPR/Cas9.
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DOI：
10.1093/nsr/nwy156
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发表时间：
2019-05
影响因子：
20.6
通讯作者：
Long C
中科院分区：
文献类型：
--
作者：
Long C
目前的教条是以无模板方式修复的Cas9产生的双链断裂导致不精确的插入/缺失（indel）突变，并且只有模板介导的同源性定向修复（HDR）可以促进精确的修饰。因此，非同源末端连接（NHEJ）的修复产物被认为在其结果上是异质的，并且通常被认为是随机的。基于对NHEJ的这种理解，它长期以来仅用于不精确的基因破坏，而不是作为基因组编辑的精确和可预测的手段。还通常接受的是，SpCas 9-一种已被广泛详细研究的变体-切割基因组靶链并产生平端。Wu和他的团队[1]打破了这两个先前接受的教条，首先证明SpCas 9介导的无模板核苷酸插入是精确和可预测的，其次令人信服地证明SpCas 9也可以使用配对指导核糖核酸（RNA）方法在5 '端产生1- 3-nt突出端的交错末端。这些发现被其他使用高通量机器学习模型的研究小组独立证实[2，3]，并建立了一个新的视角，为设计更广泛的基因组编辑策略提供了信息，该策略基于对致病性突变的可预测和无模板校正。由于Cas9介导的编辑的活动和结果以前被认为是不可预测的，大多数研究人员通常会设计和测试靶向同一基因座的多个向导RNA。只有当他们“幸运”的时候，这种方法才会产生理想的编辑结果。就像掷骰子一样，你最后肯定会得到一个“数字”，尽管不一定是赢得游戏的那个。为了-
The current dogma is that Cas9-generated double-strand breaks repaired in a template-free manner lead to imprecise insertion/deletion (indel) mutations, and that only template-mediated homology-directed repair (HDR) can facilitate precise modification. Hence, the repair products of non-homologous end joining (NHEJ) are considered heterogeneous in their outcome, and generally dismissed as random by nature. With this understanding of NHEJ, it has long been used only for imprecise gene disruption, rather than deployed as a precise and predictable means for genome editing. It is also conventionally accepted that SpCas9—a variant that has been studied in extensive detail—cleaves the genomic target strand and generates blunted ends. Wu and his team [1] break with these two previously accepted dogmas, first by demonstrating that SpCas9-mediated template-free nucleotide insertions are precise and predictable, and second by convincingly proving that SpCas9 can also generate staggered ends with 1-to 3-nt overhangs at the 5’end using a paired guide ribonucleic acid (RNAs) approach. These findings were independently confirmed by other groups using high-throughput machine learning models [2, 3] and establish a novel perspective that informs the design of a wider breadth of genome-editing strategies predicated on a predictable and template-free correction of pathogenic mutations.Since the activities and outcomes of Cas9-mediated editing were previously thought to be unpredictable, most researchers would typically design and test multiple guide RNAs targeting the same locus. Only if they got ‘lucky’would this approach yield a desirable editing outcome. Much like rolling a dice, you will most certainly end up with a ‘number’, though not necessarily the one that wins the game. To en-
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影响因子：
16.6
作者：
Nakade S;Mochida K;Kunii A;Nakamae K;Aida T;Tanaka K;Sakamoto N;Sakuma T;Yamamoto T
通讯作者：
Yamamoto T
影响因子：
16.6
作者：
Charpentier M;Khedher AHY;Menoret S;Brion A;Lamribet K;Dardillac E;Boix C;Perrouault L;Tesson L;Geny S;De Cian A;Itier JM;Anegon I;Lopez B;Giovannangeli C;Concordet JP
通讯作者：
Concordet JP
影响因子：
64.8
作者：
Shen MW;Arbab M;Hsu JY;Worstell D;Culbertson SJ;Krabbe O;Cassa CA;Liu DR;Gifford DK;Sherwood RI
通讯作者：
Sherwood RI
影响因子：
46.9
作者：
Allen, Felicity;Crepaldi, Luca;Parts, Leopold
通讯作者：
Parts, Leopold
精确且可预测的 CRISPR 染色体重排揭示了 Cas9 介导的核苷酸插入原理
影响因子：
16
作者：
Shou, Jia;Li, Jinhuan;Wu, Qiang
通讯作者：
Wu, Qiang