Department of Cell Biology and Physiology, Brigham Young University, Provo, UT, USA.
Molecular Medicine Program and Department of Neurobiology, University of Utah, Salt Lake City, UT, USA.
Nucleic Acids Res. 2021 Dec 16;49(22):e131. doi: 10.1093/nar/gkab838.
CRISPR-Cas9 sgRNA libraries have transformed functional genetic screening and have enabled several innovative methods that rely on simultaneously targeting numerous genetic loci. Such libraries could be used in a vast number of biological systems and in the development of new technologies, but library generation is hindered by the cost, time, and sequence data required for sgRNA library synthesis. Here, we describe a rapid enzymatic method for generating robust, variant-matched libraries from any source of cDNA in under 3 h. This method, which we have named SLALOM, utilizes a custom sgRNA scaffold sequence and a novel method for detaching oligonucleotides from solid supports by a strand displacing polymerase. With this method, we constructed libraries targeting the E. coli genome and the transcriptome of developing zebrafish hearts, demonstrating its ability to expand the reach of CRISPR technology and facilitate methods requiring custom libraries.
CRISPR-Cas9 sgRNA 文库改变了功能基因组筛选，还催生了几种依赖于同时靶向多个遗传位点的创新方法。此类文库可用于大量生物系统和新技术的开发，但 sgRNA 文库合成所需的成本、时间和序列数据限制了文库的生成。在这里，我们描述了一种快速酶促方法，可在 3 小时内从任何 cDNA 源生成稳健、匹配变体的文库。这种方法被我们命名为 SLALOM，它利用了定制的 sgRNA 支架序列和一种通过链置换聚合酶从固体载体上分离寡核苷酸的新方法。使用这种方法，我们构建了靶向大肠杆菌基因组和斑马鱼心脏发育转录组的文库，证明了它扩展 CRISPR 技术的能力，并为需要定制文库的方法提供了便利。
