For decades, genetic approaches including random mutagenesis, gene trapping, and gene targeting are widely used to uncover critical genes and molecular pathways in development and disease. However, the precise genome editing approach, gene targeting, through homologous recombination in mammalian cells is an extremely inefficient process. Therefore, it was not feasible to perform large-scale genetic screening at the endogenous DNA sequence level in mammalian cells with precision. In recent years, the developments of next-generations genome editing techniques including zinc-finger nuclease, transcription activator-like effector nuclease,[3,4] and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system have drastically increased the efficiency of precise genome editing by engineering a site-specific double strand DNA break (DSB). DSB induced DNA repair allows for the deletion or insertion of a DNA sequence to precisely edit the genome. Among these new methods, CRISPR/Cas9 system is arguably the most widely used genome engineering tool due to its simplicity in experimental design, high efficiency in editing, and capacity for multiplexing.
The CRISPR systems are bacterial adaptive immune systems that protect the cell against viral infection. These systems are classified into two classes: class 1 with multi-subunit effector complexes and class 2 with single-protein effector modules. There are six main types and cumulatively about 30 different subtype CRISPR systems between the two classes. The first CRISPR-based gene-editing tool was developed from the class 2 type II CRISPR system using Streptococcus pyogenes Cas9. Since then, the original CRISPR/Cas9 system has been simplified to two key components to achieve genome editing in mammalian cells: a single-guide RNA (sgRNA) and a Cas9 endonuclease. The sgRNA is a chimeric RNA with the CRISPR RNA (crRNA) and trans-activating crRNA duplexes. The Cas9 protein recognizes and binds to the trans-activating crRNA, and the crRNA functions as a guide so that Cas9 can cut the DNA target sequence to generate DSBs. The first 20 bp of the crRNA sequence can be programmed to direct Cas9 to specific genomic regions with a Protospacer Adjacent Motif (PAM) sequence. The PAM sequence allows Cas9 to load and initiate sgRNA to bind and search for the correct target site in the genome. The Cas9 proteins from different organisms have sequence specificity for PAM sequences and can be leveraged to target a variety of genomic regions[8–14] (Table 1). The versatile CRISPR/Cas9 system and its derivatives have been utilized in many different genetics applications, and in many cell types and organisms.[5,15,16] Here, we will discuss the general applications of CRISPR/Cas9-based genetic tools for genome and epigenome editing in mammalian cells, followed up by how to use these tools for high-throughput genetic screening.
Database search strategy
We performed a series of electronic searches in the PubMed database to prepare this review. For the content related to CRISPR/Cas9 based genome and epigenome editing, we used the following key terms: CRISPR, Cas9, CRISPR interference, CRISPR activation, base editing, prime editing, and CRISPR screen. We focused on publications from January 2012 to December 2019. For additional publications cited in the review, we searched with each specific keyword and did not filter for specific publication dates. The searched results were first screened by the title, abstract, and keywords. Finally, we went through the full text and extracted the relevant information from each publication.
CRISPR/Cas9-based genome and epigenome editing tools and their application in mammalian cells
CRISPR/Cas9-based genome editing results in irreversible changes in DNA sequences in two ways, non-homologous end joining and homology-directed repair (HDR), during DSB repair (Fig. 1A and Table 2). The non-homologous end joining pathway creates indel mutations or small deletions, which can cause a frame shift and disrupt the function of the gene. In contrast, the HDR pathway leads to a specific sequence change in the presence of a donor DNA with homologous sequences flanking the DSB site. CRISPR/Cas9-based genome editing has been leveraged to edit the mammalian genome, achieving over 90% indel frequency and about 30% HDR frequency.
Point mutations in the Cas9 endonuclease domains, HNH and RuvC, can inactivate Cas9 endonuclease activity and change the performance of CRISPR/Cas9-based genome editing. For instance, Cas9 nickase consists of a single inactive endonuclease domain and creates a single strand break instead of a DSB. Moreover, catalytically dead Cas9 (dCas9) consists of inactivity in both endonuclease domains and loses its cutting ability, but maintains programmed targeting.
CRISPR/Cas9 mediated HDR allows us to precisely edit the mammalian genome. However, unintended indels cause lower precise editing efficiency and leave a scar at the target site. To achieve precise genome editing at the base pair resolution, CRISPR base editing (CRISPRb) was developed to convert one nucleic acid to another without the DSB and donor template (Table 2). There are two types of base editors: cytosine base editors or adenosine base editors. These systems target specific bases by fusing different DNA modification enzymes to dCas9 or Cas9 nickase. Cytidine deaminase, such as rat cytidine deaminase APOBEC1, activation-induced cytidine deaminase, and the sea lamprey PmCDA1 gene, converts cytosine bases into uridines, and results in the conversion of a C-G pairing into a T-A[21,22] (Fig. 1Bi). This cytosine base editor, on average, reaches efficiencies of 35% in mammalian cell cultures, and an upwards of 75% depending on the sequence. DNA adenosine deaminase, an evolved Escherichia coli transfer RNA adenosine deaminase TadA, converts adenosine into inosine which is treated like guanosine by the cell (Fig. 1Bii). Therefore, this enzyme can convert an A-T pairing into a G-C. The efficiency of an adenosine deaminase base editor is about 50% in human cells. Additional proteins added into base editors can increase editing efficiency and specificity. For example, bacteriophage Mu-derived Gam protein, a nuclease inhibitor, limits indel formation. Additionally, uracil-DNA glycosylase inhibitor subverts the cellular uracil base excision repair pathway to improve base-editing efficiency and reduce indel formation. Taking advantage of base pair resolution, CRISPRb has been used in broad applications. For example, CRISPRb identifies known and novel mutations in directed gene evolution to create and improve protein function via changing the DNA codon for an amino acid. CRISPRb can also create or correct early stop codons to inactivate or correct target genes.
However, the CRISPRb tool is only able to convert four transition mutations (C→T, G→A, A→G, T→C) and, thus, cannot generate the remaining eight transversion mutations (C→A, C→G, G→C, G→T, A→C, A→T, T→A, T→G). The recently developed prime editing technology fills this gap. Prime editing uses a reverse transcriptase fused to Cas9 nickase and a prime editing guide RNA containing genetic modification information to copy genetic information from the prime editing guide RNA into the target site without generating DSBs (Fig. 1C and Table 2). The prime editing efficiency can be improved with an engineered reverse transcriptase and nicking of the non-editing strand. Compared with base editing, prime editing can directly correct pathogenic transversions. For example, prime editing directly reverted the β-globin coding gene HBB mutation (AT-to-TA transversion, the primary genetic causes of sickle cell disease) to the wild type HBB with 26–52% efficiency in HEK293T cells. Furthermore, the prime editor can generate targeted insertions and deletions without DSBs and donor DNA templates.
In addition to modifying DNA sequences, the CRISPR/Cas9 system can be used to modify the epigenome. Epigenetic status can be directly modified with dCas9 fused epigenetic modifiers, such as histone demethylation by LSD1, cytosine methylation by DNMT3A or MQ1, and cytosine demethylation by Tet1.[27,28] Furthermore, CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) approaches can modify the chromatin status to directly or indirectly modulate transcription (Table 2).
CRISPRi can be carried out either with or without a repressor domain. Without an effector domain, the dCas9 itself shows the ability of decreasing or inhibiting transcription (Fig. 1Di). When the dCas9-sgRNA complex binds to the promoter region of the target gene, the complex blocks RNA polymerase binding and, therefore, interferes with transcription initiation. Alternatively, when the dCas9-sgRNA complex binds downstream of the transcription start site of the target gene, the complex blocks RNA polymerase from moving forward and prevents transcription elongation. CRISPRi also refers to when dCas9 is fused with a repressor domain, such as the Krüppel-associated box (KRAB) domain (Fig. 1Dii). The KRAB domain recruits the KAP1/TIF1β corepressor complex. Subsequently, KAP1 acts as a scaffold to further recruit histone deacetylases, histone methyltransferases, and heterochromatin protein 1 isoforms to induce heterochromatinization at the target region for gene silencing.[30,31] The dCas9-KRAB fusion inhibits gene expression about 2 to 5 fold greater than dCas9 alone. Other effector domains, such as MeCP2, SIN3A, HDT1, MBD2B, NIPP1, and HP1A, which are associated with transcriptional regulation and gene silencing, can be fused to dCas9 to achieve stronger repression (up to 8-fold more than dCas9 alone). Bipartite fusion domains increase repression further (between 5-fold to 60-fold more than dCas9 alone). For example, the dCas9-KRAB-MeCP2 outperformed other bipartite and tripartite repressors when tested in various endogenous target genes.
In the case of CRISPRa, two types of domains are fused to dCas9 to achieve genome activation. Type I domains scaffold transcriptional factors and chromatin remodeling complexes associated with active transcription to indirectly activate transcription (Fig. 1Ei). For example, VP64 indirectly activate transcription by recruiting PC4, CBP/p300, and the SWI/SNF complex. Type II domains are usually a chromatin remodeling or modification enzyme that activate transcription. For example, the core p300 domain catalyzes acetylation of histone H3 lysine 27 at its target sites, which leads to robust transcriptional activation of target genes or distal enhancer regions (Fig. 1Eii). In general, multiple domain fusions have higher transcriptional activation efficiency than single fusions. For example, dCas9-VP64-p65-Rta achieved a 22-fold to 320-fold greater activation compared to dCas9-VP64. Therefore, several strategies have been developed to incorporate multiple activation domains including fusing a series of activation domains directly to dCas9, fusing a protein scaffold to dCas9, such as Suntag, to recruit activators, and fusing an activation domain to dCas9 and an RNA scaffold to sgRNA to recruit activation domains, as illustrated in the synergistic activation mediator system.[40,41]
CRISPR/Cas9 system mediated high throughput genetic screening
Genetics screens have led to the discovery of many genes related to biological processes. Previously, transposon mediated insertional mutagenesis and RNA interference mediated gene knockdowns were two major methods for high throughput functional screens in mammalian cells. However, transposon insertion mediated screens are limited to the transposon recognition sites, and the RNA interference mediated approach can only lead to knockdowns with the disadvantage of extensive off-target effects. CRISPR/Cas9 system mediated high throughput screens can address these limitations, and various screen methods have been developed including knock-out, knock-in, transcriptional repression, transcriptional activation,[41,47] and base editing. Both CRISPR mediated genome editing and epigenome editing can be applied to high throughput screening. The genome editing allows for the change or deletion of the DNA sequence in the genome to knock-out protein coding genes, noncoding RNAs (ncRNAs), or regulatory elements. In contrast, the epigenome editing is able to repress or activate transcription without changing the DNA sequence in mammalian cells. In addition, the base editing system pushes the screening resolution to the single base pair level. All these screening strategies can be carried out in two ways: pooled screening and single cell screening. These approaches have been applied to identify genes essential for cell viability,[44,49,50] cell fate specification and reprogramming, drug resistance, key components in biological pathways in response to drug treatment,[44,49] regulatory sequences in controlling gene regulation, and critical amino acids for protein function. Here, we will describe the general workflow for CRISPR/Cas9-mediated high throughput screening.
sgRNA library design and cloning
The first step in performing a CRISPR-based high throughput screening is the generation of a sgRNA library. Many bioinformatics CRISPR tools have been developed for designing sgRNA for various purposes and different species (Table 3). The quality of sgRNAs is typically indicated by a score, which are determined by two criteria: efficiency and specificity. Efficiency shows the likelihood of sgRNA mediated cutting, which positively associates with sgRNA GC content (the ideal GC range is 40%–80%), sgRNA stability, and DNA accessibility of the target site. Specificity accounts for the off-target sites binding of sgRNA, which is controlled by sequence similarity between the on-target site and the off-target sites. Genome-wide profiling of off-target cleavage suggests that almost all predicted mismatches of four or more in off-target sites are not cleaved by Cas9. Therefore, these kinds of predicted off-target sites do not have to be considered in sgRNA library design. In addition, two different kinds of controls, positive and negative, should be added into the sgRNA screening library in order to minimize the false positive and false negative rates of screening during the analysis. The positive control sgRNAs, typically targeting genomic regions, can affect phenotypes related to the screen such as sgRNAs against reporter genes and previously validated sgRNAs targeting essential genes linked to the screening phenotype. In contrast, negative controls can be designed to target either other genomes or genomic regions that are not related to the screening phenotype. There are published sgRNA libraries available online for researchers, which include genome-wide targeting libraries or libraries for specific purposes (https://www.addgene.org/pooled-library/#crispr). These existing libraries can be used directly for screening, but the drawbacks are that no additional modifications can be made to them directly and they may not be fit for the researcher's purpose. Usually, the libraries are constructed using pooled oligos containing designed sgRNA sequences (Fig. 2A). sgRNA libraries can be cloned into plasmids expressing either single sgRNA or dual sgRNA. For a single sgRNA library, the sgRNA pool is directly inserted into vectors via Gibson assembly or ligation. Usually, single sgRNA libraries are used to create indel mutations, or activate or inhibit transcription. In the case of a dual sgRNA library, sgRNA pairs with linkers are cloned into a sgRNA expression vector. Then, another sgRNA scaffold and U6 promoter are inserted between the sgRNAs pair to replace the linkers. Dual sgRNA expression libraries have been used for deletion screens.[51,54] Additionally, a library of sgRNA expression cassettes can be first cloned into the vector, then a library of the second sgRNA expression cassettes inserts into the vectors which already have the first sgRNA expressing cassettes. This method generates random combinations of each designed sgRNA for combinatorial screens for analyzing genetic interactions.
sgRNA library delivery
Once the designed sgRNA oligos are cloned into a desired vector, the next step is to deliver the CRISPR/Cas9 system into cells (Fig. 2B). The most common system for CRISPR/Cas9 delivery is viral delivery systems, including adeno-associated virus, lentivirus (LV) and adenovirus. Adeno-associated virus is significantly less immunogenic than other viruses, but the packaging capacity is only 4.7 kb. Therefore, Cas9 and sgRNA have to be delivered separately. In contrast, LV and adenovirus allow up to 8 kb and 35 kb packaging capacity, respectively, allowing Cas9 and sgRNA to be included within one vector. All of these viruses infect both dividing and non-dividing cells, but only LV integrates into the host genome efficiently. Adenovirus does not integrate into the host genome, and adeno-associated virus is mainly episomal. In comparison, LV can integrate into the host genome and result in stable and long-term expression of Cas9 and sgRNA. Thus, the readout of insertional sgRNA sequences in the infected cells can be detected by sequencing in later steps, making LV the most popular delivery method for high-throughput screening.
In addition to these viral delivery systems, transfection is another method for CRISPR/Cas9 system delivery. The transfection method can deliver the sgRNA library into the cell without integration into the genome.[57,58] For example, a Cas9/sgRNA library and the HDR library were co-transfected in a multiplex HDR screening. Coupling CRISPR/Cas9 mediated DSBs with multiplex HDR demonstrated a saturation of editing in genomic regions. Additionally, the transfection method can insert a sgRNA library into the genome by coupling it with another integration system, such as the transposon system. piggyBac or Sleeping Beauty transposon vectors have a greater capacity for larger insertions and have the ability of integration into the host genome. Co-transfection of transposon based sgRNA vectors and transposes were used to deliver CRISPR/Cas9 system into the cell.[57,58] One potential drawback of the transposon system is that the efficiency of transfection is lower than transduction in certain cell types.
High throughput screening
In pooled library screens, cells are infected at a very low multiplicity of infection to ensure each cell receives no more than one viral particle. To avoid dropouts during the screening, the screening library should infect cells at a high coverage (eg, 1000×). The screening can be divided into two categories, positive screening and negative screening (Fig. 2C). Positive screens are used to identify the cells which pass the selection criteria, such as cell survival or expression of reporter genes. In contrast, negative screens identify the cells which do not pass the selection criteria. After the delivery of screening library, a small portion of the infected cells are set aside to serve as a non-selected control, while the majority of the cells continue to be used for selection. In the end, the sgRNAs in these two sets of cells are polymerase chain reaction amplified and sequenced via next generation sequencing to determine which sgRNAs are enriched or depleted during selection. The enrichment or depletion of sgRNAs can be used to infer which genomic loci are essential for selection conditions. Positive screens have been used to identify genes involved in drug resistance, reveal host genes essential for Zika virus replication, and discover cis-regulatory elements. Meanwhile, negative screenings can identify essential genes for cell survival and regulatory elements which contribute to cell proliferation.[44,61]
Study coding regions with CRISPR/Cas9-mediated high throughput screening
The first genome-wide coding gene CRISPR/Cas9 screening in human cells targeted 18,080 protein coding genes and identified genes necessary for cell resistance to the treatment of vemurafenib, a therapeutic RAF inhibitor for melanoma. The reproducibility of identifying same genes with independent sgRNA targeting demonstrate the feasibility of performing genome-wide screening using CRISPR/Cas9 system. In addition to directly disrupting coding genes with random indels by sgRNA, CRISPRb enables the identification of functional domains in protein at a single amino acid resolution. Specifically, base mutations using CRISPRb can create targeted missense mutations and, thus, allow for the identification of crucial amino acids of the proteins of interest. For instance, a CRISPRb-based screening was carried out in mice for Dnd1, a critical gene for primordial germ cell development. The screen identified four amino acids that are essential for Dnd1 protein function. Missense mutations in these four amino acids can result in Dnd1 instability and thus abolish protein-protein interaction, and leads to the complete loss of Dnd1 in primordial germ cells. In parallel, epigenome screening can be used to identify genetic components important for biological processes by gene activation (CRISPRa) or repression (CRISPRi). For example, a CRISPRa screening targeting 2428 transcriptional factors and DNA binding proteins systematically identified 74 regulatory factors that positively contribute to neuronal cell fate specification. In another example, a genome wide CRISPRi screening targeting 15,977 human protein-coding genes identified genes essential for cell growth in K562 cells. The top hits from the screening were the genes involved in essential cellular processes, such as translation, transcription, and DNA replication.
Study noncoding regions with CRISPR/Cas9-mediated high throughput screening
98% of the human genome is noncoding sequences including regulatory elements, repeated elements, and ncRNAs. More than 90% of diseases or trait-associated variants identified by the genome-wide association studies are concentrated in noncoding regions.[63,64] However, the targets of noncoding regulatory elements and the precise function of ncRNAs are, for the most part, still unknown. CRISPR/Cas9 system based genetic tools have proven to be a valuable tool for annotating the noncoding region.
An ncRNA is an RNA molecule that is transcribed from genomic DNA, but not translated into a protein. Unlike protein coding genes, point mutation, small deletion, or insertion are less likely to disrupt the function of ncRNA, especially long ncRNAs (lncRNAs). For functional ablation of lncRNA, CRISPRa, CRISPRi, and CRISPR-mediated deletions have been used to study the biological function of lncRNAs with high throughput screening.[50,65,66] In addition, exon skipping or intron retention generated by targeting splice donor or splice acceptor sites with CRISPR/Cas9 system is another approach to interfere lncRNA function. A genome-scale splicing-targeted screen covering 10,996 lncRNAs was performed in chronic myeloid leukemia K562 cells and identified 230 lncRNAs that are essential for cellular growth of K562 cells. The same sgRNA library was applied in the lymphoblastoid cell line GM12878 and HeLa cell line identified 220 and 115 lncRNAs, respectively, affecting cell growth and proliferation. These screens illustrate that the approach of targeting splicing works in multiple cell lines and can be utilized to discover the diverse functions of lncRNAs.
Regulatory elements control spatial and temporal gene expression, which are crucial for organism growth, development and disease. Multiple functional assays, such as massively parallel reporter assays and self-transcribing active regulatory region sequencing, have been developed to test putative regulatory elements. However, these reporter based assays use heterogeneous promoters and cannot test DNA sequences function in its native chromatin content. To address these limitations, CRISPR/Cas9 approaches can also be applied for characterizing regulatory elements in a high throughput fashion. For example, single sgRNA libraries can be used for testing putative regulatory elements mapped by biochemical signatures with high resolution, and can even identify motifs and transcriptional factors binding sites.[70–72] On the other hand, dual-sgRNA libraries can be used to tile and delete large genomic intervals with high coverage. One example of this application is called CREST-seq, which can screen for genomic regions over megabase pairs in an unbiased manner. In addition, CRISPRi can also be applied for the discovery of regulatory elements.[61,73,74]
CRISPR/Cas9-mediated high throughput single cell screening
More recently, methods have developed to combine pooled CRISPR screenings with single-cell RNA sequencing technologies, which enable reading pooled CRISPR screens at the single-cell transcriptome resolution. Methods, including CROP-seq, Perturb-seq,[76,77] CRISP-seq, Mosaic-seq, and crisprQTL, can facilitate functional dissection of complex biological mechanisms in heterogeneous cell populations. Typically, sgRNAs are transcribed from a human or mouse U6 promoter by RNA polymerase III. Therefore, the sgRNA transcripts lacking a polyadenylated tail cannot be detected by polyadenylated enrichment during single-cell RNA sequencing. The CROP-seq construct makes the sgRNA part of the puromycin polyadenylated mRNA transcript by inserting a hU6-sgRNA cassette into a 3′ long terminal repeat so that it can be detectable by the polyadenylated enrichment RNA-seq approach. Perturb-seq and CRISP-seq use guide barcodes to detect sgRNA within individual cells. Compared to different sgRNA barcoding strategies, CROP-seq is only suitable for single perturbation in individual cells, whereas Perturb-seq and CRISP-seq allow inductions of multiple perturbations within the same cell. Therefore, Perturb-seq and CRISP-seq are useful for systematic identification of complex genetic interactions with single-cell resolution. In terms of testing non-coding regulatory elements, Mosaic-seq and crisprQTL can facilitate large-scale examination of regulatory element activity by introducing random combinations of CRISPRi mediated perturbations in each cell, followed by single cell RNA-seq.[73,74]
The CRISPR/Cas9 genome editing tools have significantly improved our ability to manipulate the genome and have revolutionized both basic and translational research. The combination of single cell technology and CRISPR system-mediated high-throughput screening in primary cells or tissue will provide an efficient approach to decoding genetic mechanisms and to develop new treatment for human diseases. In addition, since many players are involved in biological processes and work in a network with one another, expanding the current analysis to combinational screens for genetics interactions between different loci will reveal novel biological insights in human health and diseases.
One other promising application of CRISPR is therapeutic editing in disease-relevant somatic cells. There are already several preliminary examples of therapeutic interventions in model organisms and cell culture systems. For example, haploinsufficiency induced obesity can be rescued by CRISPRa mediated up-regulation of Sim1 functional allele in a Sim1 and Mc4r heterozygous mouse model. In another study, dCas9-Tet1 mediated demethylation of CGG repeats upstream of FMR1 promoter in fragile X syndrome iPSCs and neurons induced local chromatin state change and restored FMR1 expression. These examples are just the beginning towards leveraging CRISPR for developing therapeutic approaches for human diseases.
XR and MAT wrote the manuscript and prepared the figures. YS revised the manuscript. All authors reviewed and approved the final version of the manuscript.
Conflicts of interest
The authors declare that they have no conflicts of interest.
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