CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.
The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.
Recent advances in genome engineering technology have allowed for unprecedented opportunities for site-specific modification of the genome. This technology may be utilized to investigate the function of genes and regulatory elements via prospective genetic perturbation. Zinc finger nucleases (ZFNs), transcription-activator like (TAL) effector nucleases (TALENs), and CRISPR/Cas9 RNA-guided nucleases each leverage customizable DNA specificity to localize a nuclease for the introduction of DSBs1–3. The resulting DSBs can be repaired by indel-forming NHEJ or by homology-directed repair (HDR) using a donor template4.
The CRISPR/Cas9 nuclease pathway, an adaptive immune system in prokaryotic cells5, has been recently adapted for mammalian genome engineering2,3. This tool has been demonstrated to be an inexpensive, efficient, and reliable genome engineering technique6. Briefly, a complex of Streptococcus pyogenes-derived Cas9 nuclease and a sgRNA achieve target recognition via Watson-Crick base-pairing with cognate genomic DNA sequences. sgRNAs include 20-mer sequences complementary to genomic sequences adjacent to an obligate protospacer adjacent motif (PAM) NGG. Cas9 induces a DSB at a predictable position within the target site. Additionally, variants of Cas9 with single-strand cleavage capacity or catalytic inactivity may be used to facilitate “nicking” or transcriptional regulation respectively7–9. CRISPR/Cas9 has been used for a wide range of applications including both knock-in and knockout10,11, large-scale genomic deletions12–14, pooled library screening for gene discovery15,16, genetic engineering of numerous model organisms10,11,17–21, as well as gene therapy22,23.
Here we describe a protocol for efficient deletion of desired genomic regions. The protocol includes CRISPR design, cloning, and delivery, as well as deletion, identification, and characterization. Genomic deletions can be generated by the introduction of two CRISPR sgRNAs with Cas9 to induce repair of the resultant two DSBs by NHEJ with deletion of the intervening segment. This strategy has been used to create deletions ranging from one kilobase to over one megabase12. Deletions can be informative for the study of genes and other genetic elements, either in isolation or in combination. There are several potential advantages of genomic deletions as compared to HDR or single-site small indel production. First, this method capitalizes on the high efficiency of NHEJ in many cellular contexts7. The high frequency of deletion limits the number of clones needed to be screened to identify informative clones. Deletion frequency is inversely related to deletion size. Biallelic deletion clones may be retrieved at frequencies at least as great as of probabilistic expectation12. Second, both monoallelic and biallelic deletions may be easily identified and distinguished by conventional PCR, simplifying the screening process. Strategies relying on small indels or point mutations may require RFLP, allele-specific PCR, T7EN1 cleavage assay, Sanger sequencing, RT-qPCR, or immunoblotting, which may be more laborious. Third, by removing a substantial portion of a gene or element of interest, a reliable loss-of-function allele may be obtained. In contrast, frameshift mutations in protein-coding sequences may not always induce nonsense-mediated decay, may produce a hypomorphic or neomorphic allele, or target an exon excluded from an alternate isoform24. Finally, deletions may be particularly revealing for the study of non-coding DNA such as regulatory elements since frameshift mutations as produced by single-site indels would not be relevant25.
所述CRISPR / Cas9系统可以被用于产生一个尺寸范围的基因缺失。尽管我们已经观察到缺失的频率相对于预期缺失大小成反比变化,我们已经能够恢复的高达1 Mb缺失和缺失高达100kb的常规产率的多个等位基因缺失克隆。我们观察到在依次引入缺失的效率没有损失到的细胞系。这种策略可以用于创建组合删除许多基因和元件。获得双等位基因缺失克隆的方法可以通过估计所需的基础上删除尺寸,以获得克隆的期望数量与等位基因缺失12被筛选的克隆的最小数目可以加快。
获得单等位基因缺失的情况下等位基因缺失的概率分布的能力可能表明与功能完全丧失相关的细胞的杀伤力。低FRequency或不存在缺失可能反映了许多方案,包括转染较差,低效sgRNAs或低效PCR筛选引物(由于缺少一个阳性对照,以验证PCR引物来筛选缺失)。 GFP +细胞可以被用作一个替代转染效率(参见步骤5.2),因此,在GFP +细胞的降低可能反映转差和由此而来降低缺失效率。使用两种不同的因组对具有自主筛选引物可以帮助控制低效因组和筛选PCR引物,最大限度地获得等位基因缺失克隆的机会。细胞的GFP +细胞分选,丰富的缺失等位基因。虽然此步骤可以省略,遗漏将可能必要筛选更多的克隆,以确定那些具有单等位基因或等位基因缺失。到的程度的转染效率可被优化,我们希望被增强的基因组编辑效率。
<p c姑娘=“jove_content”>该NHEJ事件背后缺失和局部修补结果的一系列等位基因与各种插入缺失的在目标位点。主要的结果是小〜1 – 10碱基对的插入或更常见缺失在因组定向裂解( 图2B)的部位。通常,这些等位基因似乎是microhomology基于修34,35的结果。应当指出的是,我们描述了基于PCR的检测策略将无法确定更大或更复杂的插入,缺失,倒位,或重排。虽然这些事件是不常见的,我们观察到,其中既没有缺失,也没有非缺失的扩增子可被检测的克隆,并且在进一步的调查反映这些更复杂的结果。我们已经观察到广泛CRISPR / Cas9介导的从单等位基因和非缺失克隆的非缺失等位基因“疤痕”( 见图2B)。这些“疤痕”是由在因组切割位点不期望的缺失产生小插入缺失( 即,缺失sgRNAs A和B之间的中间段)。这些伤痕常常中断了因组目标识别。因此,我们要敦促谨慎重新目标细胞等位基因此前曝光过使用相同的sgRNAs sgRNAs。一个更成功的重新定位策略将利用独特的序列因组从之前“伤痕累累”的识别位点不同的。在情况下,当一对sgRNAs的识别外显子序列( 图1B,下图),移码等位基因可以甚至在没有缺失的产生。因此,单等位基因缺失克隆可以富集丢失的功能因移码突变在非缺失等位基因12的高频率。
一个关注与CRISPR / Cas9系统是脱靶效应, 即,基因组修饰在非预期位点36 – 38。 ř最近几个报告表明,较短的导的RNA用17 – 19个核苷酸可以减少CRISPR的频率/ Cas9基脱靶效应39。此外,使用每个目标部位2导板与一个切口酶双切口策略可以被用来创建双链断裂,同时尽量减少脱靶效应7。可替代地,类似于用于RNAi的策略,我们建议,不同对sgRNAs与非重叠protospacer序列可用于证明,所观察到的表型是在目标CRISPR / Cas9修改的结果,而不是潜在的脱靶效果。一种方便的方法是设计的至少两个相邻的,但不重叠的因组对,使得可用于多种因组对( 图1A)一套单一的筛选的引物(参见步骤2)。此外,通过重新引入丢失的序列和/或破坏的基因互补的缺失的细胞系可以证实之间的因果关系一个给定的基因缺失和表型。
对于生物学家与细胞模型系统的工作,RNAi技术代表了一个强大的工具,功能基因组学。但是,这种方法的局限性已包括不完整的减少靶mRNA转录水平,靶向相同基因的独立的试剂的效果的异质性,和已知的脱靶效应,包括基于种子和非种子的影响40 – 42。基因组编辑的战略承诺,以解决许多这些关切,并代表了未来的遗传扰动8,36,37一个令人兴奋的,互补的办法。此外,基因组编辑允许的非编码的遗传元件在不通过RNAi可能的,并且通过常规的定位具有挑战性的方式接近25的研究。我们鼓励CRISPR / Cas9代基因缺失的作为一个强大的和具体的方法,以产生和表征丧失功能的等位基因。
The authors have nothing to disclose.
Thanks to Jason Wright for suggesting the Golden Gate Assembly cloning strategy and Katherine Helming and members of Orkin lab, particularly Jian Xu, Guoji Guo, Elenoe Smith, and Partha Das for helpful discussions. This work was supported by NIH R01HL032259 and P30DK049216 (Center of Excellence in Molecular Hematology) to S.H.O. and NIDDK K08DK093705 to D.E.B.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
T4 Polynucleotide Kinase | New England Biolabs | M0201S | |
T4 DNA Ligase (with associated ligation buffer) | New England Biolabs | M0202T | |
Adenosine 5'-Triphosphate (ATP) | New England Biolabs | P0756S | |
BSA, Molecular Biology Grade | New England Biolabs | B9000S | |
BbsI Restriction Enzyme (with associated NEB Buffer 2.1) | New England Biolabs | R0539S | |
pSpCas9(BB) (pX330) | Addgene | 42230 | |
S.O.C. Medium | Life Technologies | 15544-034 | |
BTX ECM 830 | Harvard Apparatus | 45-0052 | |
BTX Solution and 2mm Cuvettes | Harvard Apparatus | 45-0803 | |
pmaxGFP Plasmid | Lonza | VPA-1003 | |
QuickExtract DNA Extraction Solution | Epicentre | QE09050 | |
HotStarTaq PCR Master Mix Kit | Qiagen | 203443 | |
Zero Blunt PCR Cloning Kit | Life Technologies | K2700-20 |