Ng Henry, Dean Neta
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA.
mSphere. 2017 Apr 19;2(2). doi: 10.1128/mSphere.00385-16. eCollection 2017 Mar-Apr.
The clustered regularly interspaced short palindromic repeat system with CRISPR-associated protein 9 nuclease (CRISPR/Cas9) has emerged as a versatile tool for genome editing in . Mounting evidence from other model systems suggests that the intracellular levels of single guide RNA (sgRNA) limit the efficiency of Cas9-dependent DNA cleavage. Here, we tested this idea and describe a new means of sgRNA delivery that improves previously described methods by ~10-fold. The efficiency of Cas9/sgRNA-dependent cleavage and repair of a single-copy yeast enhanced monomeric red fluorescent protein () gene was measured as a function of various parameters that are hypothesized to affect sgRNA accumulation, including transcriptional and posttranscriptional processing. We analyzed different promoters (, , and ), as well as different posttranscriptional RNA processing schemes that serve to generate or stabilize mature sgRNA with precise 5' and 3' ends. We compared the effects of flanking sgRNA with self-cleaving ribozymes or by tRNA, which is processed by endogenous RNases. These studies demonstrated that sgRNA flanked by a 5' tRNA and transcribed by a strong RNA polymerase II promoter increased Cas9-dependent mutations by 10-fold. Examination of double-strand-break (DSB) repair in strains hemizygous for demonstrated that both homology-directed and nonhomologous end-joining pathways were used to repair breaks. Together, these results support the model that gRNA expression can be rate limiting for efficient CRISPR/Cas mutagenesis in . is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because is genetically intractable. The recent development of CRISPR/Cas in (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in . Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in .
成簇规律间隔短回文重复序列系统与CRISPR相关蛋白9核酸酶（CRISPR/Cas9）已成为一种用于[具体物种]基因组编辑的通用工具。来自其他模型系统的越来越多的证据表明，单导向RNA（sgRNA）的细胞内水平限制了Cas9依赖性DNA切割的效率。在此，我们验证了这一观点，并描述了一种新的sgRNA递送方法，该方法将先前描述的方法改进了约10倍。测量了单拷贝酵母增强型单体红色荧光蛋白（[具体蛋白名称]）基因的Cas9/sgRNA依赖性切割和修复效率，作为假设影响sgRNA积累的各种参数的函数，包括转录和转录后加工。我们分析了不同的启动子（[具体启动子名称]、[具体启动子名称]和[具体启动子名称]），以及不同的转录后RNA加工方案，这些方案用于生成或稳定具有精确5'和3'末端的成熟sgRNA。我们比较了用自我切割核酶或经内源性核糖核酸酶加工的tRNA侧翼连接sgRNA的效果。这些研究表明，由5'tRNA侧翼连接并由强RNA聚合酶II启动子转录的sgRNA使Cas9依赖性[具体突变类型]突变增加了10倍。对[具体基因名称]半合子菌株中双链断裂（DSB）修复的检查表明，同源定向和非同源末端连接途径均用于修复断裂。总之，这些结果支持这样一种模型，即gRNA表达可能是[具体物种]中高效CRISPR/Cas诱变的限速因素。[具体物种名称]是一种重要的人类真菌病原体。由于[具体物种名称]在遗传上难以处理，对真菌毒力因子的理解一直很缓慢。[具体物种名称]中CRISPR/Cas的最新发展（V.K.Vyas、M.I.Barrasa、G.R.Fink，《科学进展》1：e1500248，2015，https://doi.org/10.1126/sciadv.1500248）有可能规避这个问题。然而，正如在其他生物体中发现的那样，CRISPR/Cas诱变效率可能会令人沮丧地变化。在此，我们系统地研究了假设改变sgRNA细胞内水平的参数，以优化[具体物种名称]中的CRISPR/Cas。我们最重要的结论是，增加sgRNA表达和成熟显著提高了[具体物种名称]中CRISPR/Cas诱变效率约10倍。因此，我们预计此处描述的修饰将进一步推动CRISPR/Cas在[具体物种名称]基因组编辑中的应用。
