enAsCas12a Enables CRISPR-Directed Evolution to Screen for Functional Drug Resistance Mutations in Sequences Inaccessible to SpCas9.
enAsCas12a Enables CRISPR-Directed Evolution to Screen for Functional Drug Resistance Mutations in Sequences Inaccessible to SpCas9.
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DOI：
10.1016/j.ymthe.2020.09.025
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发表时间：
2021-01-06
期刊：
影响因子：
--
通讯作者：
Daelemans D
中科院分区：
文献类型：
--
作者：
Neggers JE;Jacquemyn M;Dierckx T;Kleinstiver BP;Thibaut HJ;Daelemans D
虽然耐药突变为药物靶点接合提供了金标准证据，但从表型筛选中识别的抑制剂的靶向去卷积仍然具有挑战性。通过在蛋白质编码序列上平铺CRISPR-Cas核酸酶来遗传筛选功能性框内耐药性突变是一种用于鉴定药物的靶标和结合位点的方法。然而，这种方法的适用性受到跨基因区域的核酸酶靶位点的可用性的限制，所述基因区域在突变时介导耐药性。在这项研究中，我们发现了一种增强的AsCas 12 a变体（enAsCas 12 a），它具有更大的靶向范围，有助于筛选耐药突变，在其他CRISPR核酸酶（包括原型SpCas 9）无法访问的区域中具有更高的活性和分辨率。利用enAsCas 12 a，我们发现了针对NAMPT和KIF 11抑制剂的新的耐药突变。这些发现表明，enAsCas 12 a是CRISPR筛选工具箱中一个有前途的新成员，可以靶向SpCas 9不易接近的位点。跨蛋白质编码序列的CRISPR-Cas诱变筛选允许药物靶标去卷积。然而，由于缺乏CRISPR靶位点，这种方法的适用性受到严重限制。在这项研究中，我们发现增强的AsCas 12 a（enAsCas 12 a）CRISPR内切核酸酶具有扩大的靶向范围，有助于有效筛选耐药突变。
While drug resistance mutations provide the gold standard proof for drug target engagement, target deconvolution of inhibitors identified from a phenotypic screen remains challenging. Genetic screening for functional in-frame drug resistance mutations by tiling CRISPR-Cas nucleases across protein coding sequences is a method for identifying a drug’s target and binding site. However, the applicability of this approach is constrained by the availability of nuclease target sites across genetic regions that mediate drug resistance upon mutation. In this study, we show that an enhanced AsCas12a variant (enAsCas12a), which harbors an expanded targeting range, facilitates screening for drug resistance mutations with increased activity and resolution in regions that are not accessible to other CRISPR nucleases, including the prototypical SpCas9. Utilizing enAsCas12a, we uncover new drug resistance mutations against inhibitors of NAMPT and KIF11. These findings demonstrate that enAsCas12a is a promising new addition to the CRISPR screening toolbox and allows targeting sites not readily accessible to SpCas9. CRISPR-Cas mutagenesis screening across protein coding sequences allows for drug target deconvolution. However, the applicability of this approach can be severely restricted by the absence of CRISPR target sites. In this study, we show that the enhanced AsCas12a (enAsCas12a) CRISPR endonuclease, which harbors an expanded targeting range, facilitates efficient screening for drug resistance mutations.
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