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0Accepted Manuscript
Title: Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing
Author: Melissa L. Kelley Zaklina Strezoska Kaizhang He Annaleen Vermeulen Anja van Brabant Smith
PII: DOI:
Reference:
S0168-1656(16)31355-4
http://dx.doi.Org/doi:10.1016/j.jbiotec.2016.06.011 BIOTEC 7589
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Journal of Biotechnology
Received date: Revised date: Accepted date:
27-4-2016 31-5-2016 10-6-2016
Please cite this article as: Kelley, Melissa L., Strezoska, Zaklina, He, Kaizhang, Vermeulen, Annaleen, Smith, Anja van Brabant, Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing.Journal of Biotechnology http://dx.doi.org/10.1016Zj.jbiotec.2016.06.011
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Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing
Melissa L. Kelley, Zaklina Strezoska, Kaizhang He, Annaleen Vermeulen, Anja van Brabant Smith* Dharmacon, part of GE Healthcare, Lafayette, CO, 80026, USA * To whom correspondence should be addressed. Email: anja.smith@ge.com ABSTRACT
The CRISPR-Cas9 system has become the most popular and efficient method for genome engineering in mammalian cells. The Streptococcus pyogenes Cas9 nuclease can function with two types of guide RNAs: the native dual crRNA and tracrRNA (crRNA:tracrRNA) or a chimeric single guide RNA (sgRNA). Although sgRNAs expressed from a DNA vector are predominant in the literature, guide RNAs can be rapidly generated by chemical synthesis and provide equivalent functionality in gene editing experiments. This review highlights the attributes and advantages of chemically synthesized guide RNAs including the incorporation of chemical modifications to enhance gene editing efficiencies in certain applications. The use of synthetic guide RNAs is also uniquely suited to genome-scale high throughput arrayed screening, particularly when using complex phenotypic assays for functional genomics studies. Finally, the use of synthetic guide RNAs along with DNA-free sources of Cas9 (mRNA or protein) allows for transient CRISPR-Cas9 presence in the cell, thereby resulting in a decreased probability of off-target events.
Keywords
RNA synthesis; gene editing; synthetic crRNA; synthetic tracrRNA; dual RNA 1. INTRODUCTION
The clustered regularly interspaced palindromic repeats (CRISPR)-associated protein 9 (Cas9) system is an RNA-guided defense mechanism in bacteria and archaea. In these organisms, the Cas9 nuclease associates with two RNAs, the CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA), to direct sequence-specific cleavage of foreign DNA. This bacterial acquired immune system has been shown to be effective for gene editing in mammalian cells, and is now routinely used as an effective genome engineering tool in multiple organisms (1-4). The most commonly used system in mammalian cell applications is derived from the Streptococcus pyogenes Type II CRISPR-Cas9 system and consists of the Cas9 protein as the nuclease that cleaves double-strand DNA when guided by the crRNA and tracrRNA dual RNA (Figure 1a). Upon site-specific double-strand DNA cleavage, mammalian cells repair the break through multiple DNA double-strand break (DSB) repair pathways; the predominant pathways are non-homologous end joining (NHEJ) which results in imprecise insertions and deletions (indels), and
homology-directed repair (HDR) which results in precise genome editing when a donor template is available.
In order to facilitate expression of the RNAs, Jinek et al. demonstrated that the crRNA and tracrRNA could be expressed as a single RNA molecule resulting in effective gene editing in mammalian cells (1). This chimeric single guide RNA (sgRNA) combines the crRNA and the tracrRNA into a continuous sequence attached by a loop (Figure 1 b) and has comparable functionality to the native dual RNA system (Cas9:crRNA:tracrRNA) (1). The use of the chimeric sgRNA facilitates the cloning and expression of the two small RNAs as a single transcript for expression vectors, constructs for in vitro transcription, and viral vectors for pooled library screening. Given the ease of cloning and expressing a single guide RNA molecule rather than the crRNA and the tracrRNA separately, it is not surprising that the majority of publications thus far have utilized the CRISPR-Cas9 system with an expressed chimeric sgRNA. Nonetheless, our data ((5) and Figure 1c) as well as data from recent publications (6-8) have confirmed the original Jinek et al. findings, demonstrating that the native, dual RNA complex functions similarly to the sgRNA. For the purposes of this review, we will refer to the RNA component of the CRISPR-Cas9 system as the guide RNA (gRNA), which may consist of either the chimeric single guide RNA (sgRNA) or the dual RNAs (crRNA:tracrRNA).
2. CHEMICAL SYNTHESIS OF THE GUIDE RNA
The RNA components of the CRISPR-Cas9 system can be generated enzymatically or through chemical synthesis. Enzymatic synthesis of the gRNA by in vitro transcription utilizes T7, T3, or SP6 RNA polymerase in the presence of ribonucleoside triphosphates and a DNA template, and is a cost-effective method for generating unmodified RNAs of different lengths, including generation of sgRNAs (9). However, the method requires sequencing of the DNA template for accuracy, removal of unincorporated triphosphates, and purification of protein and DNA components from the transcribed RNA. Additionally, this method can result in errors toward the 5' end of the molecule (10).
Chemical generation of gRNAs utilizes solid-phase synthesis with nucleoside phosphoramidite building blocks to construct the gRNA. This method has the flexibility to quickly and accurately generate different sequences and lengths of RNA without the need for multiple cloning and sequencing steps. Several different types of solid-phase RNA synthesis chemistries are available including 2'-silyl (2'-TBDMS, 2'-TOM), 2'-O-thionocarbamate (TC) (11) and 2'-bis(acetoxyethoxy)-methyl ether (2'-ACE) (1214). Chemical synthesis of gRNAs has been used to successfully generate gRNA sequences for S. pyogenes Cas9 systems (5) and can be used to generate RNAs for programming of alternate Cas9 nucleases, species, or CRISPR-Cas systems (3,15-18). It is important to note that with traditional chemistries, such as TBDMS or 2'-TOM (19,20), it can be challenging to accurately and efficiently synthesize RNA greater than ~ 70 bases. For S. pyogenes, the crRNA, tracrRNA and sgRNA are
approximately 40, 70 and 100 nucleotides in length, respectively (1), which would make synthesis of the tracrRNA or an sgRNA challenging for most chemistries. Using 2'-ACE or TC chemistries (11,13), long RNA (up to 150 nucleotides) can be routinely synthesized with faster coupling rates, higher yields and greater purity than any other RNA chemistries, thus making these synthesis methods ideal for tracrRNA and sgRNA syntheses.
The relatively shorter length of the S. pyogenes Cas9 nuclease crRNA (~ 40 nucleotides) allows for high throughput chemical synthesis and rapid generation of crRNAs in large numbers. Using this approach, arrayed genome-scale collections of gene engineering reagents can be easily produced and used for large-scale functional genomics studies as discussed in more detail below. The ~ 70 nucleotide tracrRNA can be synthesized and purified in bulk for use with the target-specific crRNA. Synthetic crRNA:tracrRNAs is compatible with a variety of delivery methods including lipid transfection, electroporation, and microinjection and are therefore suitable for a wide variety of gene editing applications when combined with a source of Cas9 (Figure 2a). As shown in Figure 2b, the synthetic crRNA:tracrRNA reagents can be co-delivered with a plasmid expressing Cas9, Cas9 mRNA, or Cas9 protein, or can be delivered on their own into cells that are stably expressing Cas9 (Figure 2c). In all cases, genome editing is achieved, as indicated by the formation of indels at the site of the CRISPR-Cas9-mediated double-strand DNA break.
If a synthetic chimeric single guide RNA (sgRNA) is desired, using RNA chemistries such as 2'-ACE or 2'-TC that result in accurate synthesis of such long RNAs (~ 100 nucleotides) is essential. The synthesis of long RNAs, even with ideal synthesis approaches, still requires longer synthesis times and results in lower yields compared to shorter RNA syntheses and typically requires additional purification. Therefore, the synthesis of sgRNAs will generally be achieved with a lower throughput compared to the dual RNA approach.
3. CHEMICAL MODIFICATIONS OF SYNTHETIC GUIDE RNA
Chemical synthesis is the only method that allows for the incorporation of site-specific chemical modifications in gRNA. The type and position of chemical modifications can be hypothesized based on the unique features of the CRISPR-Cas9 system as well as the lessons learned from existing RNA-based gene modulation technologies (21). Chemical modifications of RNAs for other technologies have been used for improving editing efficiency, specificity, in vivo stability, cellular delivery and evading the innate immune system (22-24).
A large body of work studying synthetic oligonucleotides has elucidated rules for chemical modifications that may be applicable to the gRNA of the CRISPR-Cas9 system (25,26). Chemical modifications can be applied to the oligonucleotide's phosphate backbone, sugar, and/or base (Figure 3). Backbone modifications such as phosphorothioates modify the charge on the phosphate backbone and aid in the delivery and nuclease resistance of the oligonucleotide (27); modifications of the sugar, such as
2'-O-methyl (2'-OMe), 2'-F, and locked nucleic acid (LNA), enhance base pairing as well as nuclease resistance (28). Chemically modified bases such as 2-thiouridine or N6-methyladenosine, among others, can allow for either stronger or weaker base pairing (29,30). Additionally, RNA is amenable to both 5' and 3' end conjugations with a variety of functional moieties including fluorescent dyes, polyethylene glycol, or proteins. A wide variety of potential modifications can be applied to a chemically synthesized RNA molecule, yet any type of chemical modification alters the fundamental oligonucleotide structure and can therefore affect any step in the CRISPR-Cas9 gene editing mechanism. For example, modifying an oligonucleotide with a 2'-OMe to improve nuclease resistance will also change the binding energy of Watson-Crick base pairing. Furthermore, a 2'-OMe modification can affect how the oligonucleotide interacts with a transfection reagent, proteins or any other molecule in the cell. Therefore, empirical testing to determine the advantages and potential disadvantages of any chemical modification on functionality of the gRNA is essential.
A few examples applying chemical modification strategies to synthetic sgRNAs for CRISPR-Cas9-based techniques have been reported to date (31-33). A synthetic RNA approach was used to optimize crRNA length, and chemical modifications were applied to both the phosphate backbone and sugar to improve resistance to nuclease digestion, hybridization to tracrRNA and pharmacokinetics (32). Modifications including phosphorothioates, 2'-OMe, 2'-F, and 2'-constrained ethyl (2'-cEt) were applied to regions of the crRNA that recognize their DNA targets or to the tracrRNA anti-repeat region and were found to increase the efficiency of gene disruption compared to unmodified crRNA. Hendel et al. tested sgRNAs with both backbone and sugar modifications that confer nuclease stability and can reduce immunostimulatory effects (31). Upon co-electroporation or sequential electroporation of Cas9 mRNA and modified synthetic sgRNAs in human primary immune cells, sgRNAs containing 2'-OMe, 2'-OMe 3'-phosphorothioate (MS), or 2'-OMe 3'-thioPACE (MSP) modifications resulted in ten times higher editing frequencies than the unmodified sgRNA. Modifications to the sgRNA also improved gene editing efficiencies (approximately three fold) when delivering the modified sgRNA complexed with the Cas9 protein as RNPs; these improvements in editing efficiencies are primarily due to enhanced nuclease stability. While chemical modifications of synthetic sgRNAs (MS and MSP modifications) improved the nuclease stability and editing efficiency in human primary T cells and CD34+ hematopoietic stem and progenitor cells, they did not provide any consistent ability to reduce off-target effects, as variable sequence-specific effects were observed (31). Chemically modified crRNAs are likely to exhibit editing-independent effects that are based on the mechanisms of oligonucleotide-based modulation tools such as antisense, siRNA, and aptamers. For example, we observed that modified crRNAs with > 50% 2'-F modifications in the DNA binding region sequence-specifically reduced gene expression without the presence of Cas9 protein, suggesting an antisense-type mechanism (data not shown). Chemically modified crRNAs may also exhibit effects such as cellular toxicity and innate immune response due to the structure of the modified oligonucleotide. Indeed, similar to observations in siRNA and antisense
molecules (34), we observed toxicity of crRNAs with phosphorothioate chemical modifications in certain structural contexts (data not shown). While researchers in the field are using chemical modifications to reduce off-target effects and the innate immune response, studies to examine the editing-independent effects of chemically modified gRNAs are only now underway.
Similar to the observations using modified sgRNAs, we have found that chemical modifications can enhance editing efficiency by improving nuclease stability of the synthetic dual RNA system when utilizing certain delivery methods. For example, if unmodified synthetic crRNA:tracrRNAs are co-electroporated with Cas9 mRNA, editing is not detected (Figure 4a). However, if Cas9 mRNA is electroporated first and unmodified crRNA:tracrRNA is electroporated 6 hours later (sequential electroporation), efficient gene editing is observed. When both crRNA and tracrRNA are chemically modified for nuclease stability with three 2'-OMe 3'-phosphorothioates (3xMS) on the 5' and 3' ends, efficient gene editing is achieved when these modified crRNA:tracrRNAs are co-electroporated with Cas9 mRNA, similar to what was demonstrated for sgRNAs (31). MS modifications do not improve editing in sequential electroporation, suggesting that the modifications are not inherently improving the CRISPR-Cas9 gene editing mechanism, but are instead enhancing nuclease stability of the RNAs so they remain intact and are able to be loaded into Cas9 once the Cas9 mRNA is translated into protein. Chemical modifications that enhance nuclease stability are not required for lipid-mediated delivery of Cas9 mRNA and crRNA:tracrRNA and do not enhance the overall level of indel formation (Figures 1c, 2b, and 4b). This result is likely due to protection of the unmodified RNAs in the lipid complex during delivery, and rapid complexing of the Cas9 protein and the RNA resulting in DSBs (35,36).
In terms of therapeutic applications, the CRISPR-Cas9 system will likely face similar obstacles as small interfering RNA (siRNA) and antisense therapeutics due to the inherent features of the RNA: poor intracellular delivery, limited in vivo stability, unpredictable immune responses, and unwanted off-targeting effects. However, the CRISPR-Cas9 system has unique physical and mechanistic properties compared to RNAi and antisense technologies. The crystal structures of the active Cas9:gRNA ribonucleoprotein (RNP) complex show that the gRNA is bound within the large Cas9 protein and much of the RNA is not exposed to solvent (37-39). These data provide the ability to make informed choices regarding potential positions on the RNA where chemical modifications to improve therapeutic properties, such as nuclease resistance or specificity, are feasible. Furthermore, the gene editing mechanism of CRISPR-Cas9 results in a permanent change in the genome; therefore, persistent application of the CRISPR-Cas9 molecules is not necessary and stability of the RNA may not be a requirement for therapeutics. Indeed, it may be preferable to have CRISPR-Cas9 components persist just long enough to result in the genome edit. Such transient delivery can be achieved by delivering the relatively cationic Cas9 protein and the anionic gRNA as an RNP complex in cells or in vivo (35) using either lipid-mediated delivery or electroporation (40). Evasion of the innate immune system may be less of a challenge using this approach, as exposure to immunogenic RNA is limited.
4. CRISPR-CAS9 SPECIFICITY
Whether using synthetic gRNAs or expressed sgRNAs for CRISPR-Cas9 experimentation, the desired outcome is to exclusively target the intended genomic location and avoid any editing events in other regions of the genome. Several early studies demonstrated that the nuclease activity of Cas9 can be triggered at target sites with imperfect complementarity between the target DNA and the gRNA sequences; these regions of imperfect complementarity included mismatches (41-43) and insertions or deletions between the target DNA and the gRNAs (44). The tolerance of mismatches and gaps/bulges (flaws) between any given gRNA and target DNA is sequence-dependent and influenced by the position, number and distribution of these flaws throughout the ~ 20 nt target sequence. Multiple studies, including ours, have shown that single mismatches are generally well-tolerated at the 5' end of the gRNA target region, but are less tolerated at the PAM proximal end (5,41,42). This result is consistent with a model in which the "seed" region complementarity drives selectivity (1,45); however, this is sequence dependent and the size of this seed region can vary from 7 to 12 nucleotides. Two adjacent mismatches are not well tolerated compared to single mismatches, but can still result in cleavage for some sequences, while three or more adjacent mismatches generally decrease or eliminate activity. In addition, Lin et al. showed that Cas9 can have off-target cleavage when DNA sequences have an extra base (DNA bulge) or a missing base (gRNA bulge) at various locations compared to the corresponding gRNA strand (44). The correlation between cleavage activity and the position of the DNA bulge or gRNA bulge relative to the PAM appeared to be GC content and sequence dependent. While both RNA and DNA bulges can lead to off-target editing, it was found that gRNAs bulges are better tolerated, therefore maintaining formation of DSBs, than DNA bulges of similar size.
While these early references on CRISPR-Cas9 specificity demonstrate off-target cleavage with varying frequencies at genomic sites based on computational predictions (41-43), the extent of off-target effects on the genome as a whole remains unclear. Some studies suggest that CRISPR-Cas9 has substantial off-targeting activity (41,46), while other studies, including whole genome sequencing of human pluripotent stem cell (PSC) and induced PSC clones (47,48) detected very few to no off-target mutations. The need to further characterize off-target effects on a genome-wide scale in an unbiased manner has resulted in a variety of whole genome mutation analysis techniques; some newly developed techniques include GUIDE-seq (49), Digenome-Seq (50), BLESS (51), HTGTS (52) and IDLV (53). Nevertheless, even with these new whole genome methods for detection of off-targets, there still are contradictory conclusions as to the prevalence of off-target effects, from low (50) to high levels of offtargeting (49); these discrepancies can be attributed to differences in the sensitivities of each method, differences in the manner in which off-targets are identified (detection of the DSBs, indels after NHEJ, or translocations), and differences in the gRNA sequences used in the studies (for a review of recent offtarget detection methods, see (54,55)).
4.1 METHODS FOR ENHANCING CRISPR-CAS9 SPECIFICITY
Tolerance for the overall level of CRISPR-Cas9 off-targeting activity will differ based on the type of genome editing application. For example, therapeutic applications will have a much lower tolerance for off-target effects compared to other research applications such as functional genomics screening where multiple reagents can and should be used to address the issue of false positive results due to offtargeting. To enhance the specificity of CRISPR-Cas9, multiple strategies for improving Cas9 specificity have been developed, including design of the gRNA, use of high-fidelity Cas9 mutants, and formats of CRISPR-Cas9 reagents.
The most straightforward approach for improving reagent specificity is to computationally predict off-targets and avoid those sequences when designing a gRNA. Many computationally predicted offtarget sites have been experimentally confirmed, thereby supporting the validity of the computational approach. However, not all actual off-target sites have been predicted using this approach, including some off-targets with multiple mismatches. Most extant gRNA specificity evaluation tools are based solely on mismatch checking (56-59) while some actual off-targets can be explained by the presence of gaps rather than mismatches between the gRNA and the target DNA (44); this finding highlights the need for analysis tools that incorporate not only mismatches but also gaps on gRNA and DNA target sites. Recent advances have been made to incorporate these features for a more comprehensive off-target analysis, specifically in the COSMID tool (60) as well as the Dharmacon CRISPR specificity tool (http://dharmacon.gelifesciences.com/tools-and-calculators/crispr-specificity-tool/; Lenger et al., in preparation).
The structure of the gRNA or the Cas9 protein can also be altered to improve specificity. Truncation of the gRNA on the 5' end from 20 nt to 18 or 17 nt has been shown to preserve nearly all on-target cleavage activity while reducing activity at off-target sites (61,62). Using two extra G nucleotides at the 5' end of the gRNA (GGX20) has also been reported to improve the Cas9 nuclease precision (63,64). Studies on larger sequence sets are necessary to determine the universality and practical benefits of these alterations, and to identify additional sequence positions or structural changes for increased specificity. Multiple approaches that involve mutations of the Cas9 protein itself have been described with substantial improvement to the Cas9 nuclease specificity. A Cas9 mutant (Cas9 D10A) that transforms the enzyme from a endonuclease that cuts both strands to a single-strand nickase has been successfully used with paired sgRNAs to introduce nicks in close proximity to both strands (2,64-66). While this has been shown to reduce off-targeting, the need for appropriately positioned and oriented paired gRNAs can be technically challenging, especially for genome-scale library-based applications. Recently, two groups reported Cas9 variants with increased specificity by modifying amino acids in the Cas9 protein that are presumably involved in DNA stabilization: SpCas9-HF1 (a high-fidelity variant (18)) and eSpCas9 (enhanced specificity Cas9 variant (67)). Both groups showed increased specificity as demonstrated by
whole genome mutation methods. This approach of mutating non-specific DNA contacts could be additionally extended to other CRISPR nucleases.
Off-targeting can also be ameliorated by controlling the expression level or persistence of gRNA and/or Cas9 nuclease. Whole genome off-target analyses to date have been performed using expressed Cas9 and expressed sgRNAs (49,51,53,63,68). However, continuous expression of Cas9 and sgRNA in cells can lead to accumulation of off-targeting. Indeed, constitutive expression of lentiviral-based Cas9 and sgRNAs for pooled screening purposes lead to an enrichment of sgRNA constructs targeting predicted off-target sites over time (69,70), while reducing the concentration of delivered plasmid during transfection was shown to decrease off-targeting (42,53). These data support the idea that controlling the expression of Cas9 and the gRNAs in order to limit the time of action can further reduce genome-wide offtargeting.
Synthetic gRNAs can be utilized in CRISPR-Cas9 experiments to provide a unique solution for avoidance of long-term expression that would lead to off-target effects. Our analysis of mismatch tolerance upon delivery of synthetic crRNA:tracrRNA in Cas9-expressing cells showed that less than 4% of gRNA sequences containing two base mismatches to the target DNA remain active (5); this is a lower rate than observed in prior studies using expressed sgRNAs in which they found a hit rate of 6-26% of mismatched off-target sites (41,42). Additionally, studies using gRNAs with recombinant Cas9 nuclease as a ribonucleoprotein (RNP) complex delivered into cells by electroporation have demonstrated substantial decreases in off-targeting while maintaining high on-target efficiency (31,36,71,72). The gRNA:Cas9 RNPs were shown to cleave the target chromosomal DNA between 12 and 24 hours after delivery and the frequency of gene editing reached a plateau after one day; for plasmid expression of Cas9 and the gRNA, equivalent gene editing levels were only achieved at three days after delivery (36). Furthermore, the Cas9 protein has been shown to be degraded rapidly in cells, within 24-48 hours after delivery, compared to several days when continuously expressed from a plasmid (36,72). Kim et al. showed efficient RNP delivery in human fibroblasts and pluripotent cells and a reduction in off-target mutations obtained with plasmid transfection at off-target sites that differed by one or two nucleotides from on-target sites (36). The ratio of the indel frequency at the on-target site to the off-target sites for two gRNA decreased from 114 and 279 for plasmid-expressed Cas9, to 12 and 22 for Cas9 RNP delivery, indicating more than ten-fold higher specificity. These results are in agreement with those observed using direct delivery of ZFNs, which reduces off-target effects associated with plasmid delivery (73). Using in vitro transcribed gRNA and/or in vitro transcribed Cas9 mRNA will also limit the expression and persistence of CRISPR-Cas9 reagents for lower off-target effects.
Taken together, gRNAs should be designed to avoid known and predicted off-target sites, and may also be designed with variations in the length or sequence composition of the gRNA. Well-designed gRNAs can be used in conjunction with mutated Cas9 nucleases to avoid off-targeting. Importantly, a DNA-free approach, such as using synthetic crRNA:tracrRNA and Cas9 protein or Cas9 mRNA, can
further control the expression level and duration of activity of the both the gRNA and Cas9 for increased specificity.
5. GENE EDITING APPLICATIONS USING SYNTHETIC GUIDE RNAS
With the variety of reagent formats available for gRNAs (chemically synthesized, expressed, in vitro transcribed) and Cas9 (protein, mRNA, expressed), the CRISPR-Cas9 system can be suited for many distinct genome engineering applications. Vector-based CRISPR-Cas9 systems require cloning and sequence verification of each sgRNA vector which can be laborious, especially if the goal is studying tens or thousands of gene targets. Likewise, in vitro transcribed sgRNAs also require additional time and quality control to ensure consistency in length and purity of the transcribed gRNA. In contrast, chemical synthesis can easily be employed for rapid generation of the crRNA and tracrRNA molecules or the synthetic sgRNA for direct delivery into cells. This approach enables applications unique to synthetic gRNAs including arrayed screening for gene knockout studies with a variety of phenotypic readouts and at whole-genome scale as well as DNA-free methods for clonal cell line development.
5.1 GENOME-SCALE SCREENING APPLICATIONS
Shortly after the discovery of the CRISPR-Cas9 system for gene editing in mammalian cells, vector-based sgRNA pools were generated and used to perform genome-scale loss-of-function screens in a pooled format (69,70). Despite their power as a functional genomics tool, pooled sgRNA screens are limited by the types of assays that are amenable to readouts in a population of cells; specifically, the phenotypes must be selectable or sortable in order to distinguish hits from non-hits in the cell population. The ability to perform CRISPR-Cas9 loss-of-function screens in an arrayed, well-by-well fashion expands the types of phenotypic readouts that can be used. For example, arrayed screens can be performed using highcontent and morphology-based assays, and can include the measurement of multiple parameters either in an end-point or a kinetic fashion, using time lapse images, for example. Vector-based arrayed screening reagents are available but have associated challenges with the number of steps necessary to prepare the delivery reagents for screening, which can impact data quality and lead to biased results; these challenges include variability in expression levels of the sgRNA and/or Cas9 nuclease and well-to-well variability in transfection of plasmid components or the titer of packaged lentiviral particles. Chemical synthesis of gRNAs for CRISPR-Cas9 gene editing, on the other hand, allows for accurate and rapid production of arrayed gRNA libraries with high well-to-well consistencies from reagent purity and normalized concentrations.
As a first step for a generalized arrayed screening workflow using synthetic crRNA:tracrRNA, we recommend generating Cas9-expressing stable cell lines if possible. Generation of Cas9 stable cell lines enables higher phenotypic penetrance and simplifies the delivery of the synthetic gRNAs for arrayed screening (Figure 5a). We have shown that knockout of proteasome-related genes by high-throughput transfection of synthetic crRNA:tracrRNA in a U2OS Ubi[G76V]-EGFP reporter cell line lead to an
increase of EGFP signal, demonstrating that arrayed synthetic gRNAs can be used to perform functional gene knockout screens (5). While the phenotype could be observed when synthetic crRNA:tracrRNA are co-delivered with Cas9 expression plasmid (data not shown), we find that the efficiency of editing and the phenotypic response was higher and more consistent when the synthetic gRNAs are delivered into U2OS Ubi[G76V]-EGFP cells that stably express Cas9 (Figure 5b).
We have since performed several additional phenotypic readouts using Cas9-expressing cells, including a homogenous Caspase-3/7 enzymatic assay. Transfection of Cas9-expressing U2OS cells with synthetic crRNA:tracrRNAs targeting sixteen genes identified three genes (PLK1, KIF11 and BCL2L1) that lead to increased apoptosis, as measured by the Apo-ONE® Caspase-3/7 homogeneous assay (Promega) at 72 hours post-transfection (Figure 5c). In addition, transfection with synthetic crRNA:tracrRNAs targeting PLK1 and KIF11 genes that play role in mitosis resulted in a decrease in overall cell number and a 5- to 7-fold increase in mitotic index at 48 hours post-transfection as measured by the percent of cells stained for phospho-histone H3 using a high content analysis system (Figure 5d). These assays demonstrate that efficient phenotypic penetrance is elicited by the CRISPR-Cas9 system upon delivery of synthetic crRNA:tracrRNA molecules. The assays we used were all performed in a cell population using a short-term assay without the need for enrichment using cell sorting or antibiotic selection. Given the ease of transfecting RNAs into most cell types, as well as the ability to create and use Cas9-expressing stable cell lines, many phenotypic assays are expected to be amenable to arrayed screening using synthetic crRNA:tracrRNA.
For cell types where generation of Cas9-stable lines is not feasible (e.g., primary cells), the synthetic crRNA:tracrRNAs could be efficiently co-delivered with Cas9 mRNA or Cas9 protein, although this would certainly add to the transfection optimization and cost of the screening. Similarly, for cells resistant to lipid-based transfection reagents, electroporation of synthetic gRNAs with Cas9 mRNA or Cas9 protein could be used. With the recent availability of arrayed collections of predesigned synthetic crRNAs for high-throughput gene editing studies across gene families (Dharmacon, part of GE Healthcare), we anticipate a significant number of arrayed knockout screens using synthetic gRNAs to be published in the near future (74).
5.2 DNA-FREE GENOME ENGINEERING APPLICATIONS
Because gene editing results in a permanent change in the genome, once the gene editing event has occurred, it is no longer necessary for the CRISPR-Cas9 reagents to be expressed or persist in the cell. As discussed above, there may be disadvantages to having active gene editing reagents remain in cells, such as an increase in generation of non-specific DSBs in the genome (41,43,44,46,49,61,64,75). While off-target effects may be less of a concern in screening applications since any identified "hits" will be confirmed through follow up experiments, constitutive expression or high stability of the Cas9 nuclease
and/or the gRNA may be undesirable for many applications, such as generation of clonal cell lines for perpetual studies of knockout of a specific gene.
In addition to the increased potential for off-target effects due to constitutive expression of components of the CRISPR-Cas9 system, we (unpublished), and others, have found incorporation of portions of plasmid sequence used to deliver and express the CRISPR-Cas9 components during the NHEJ repair process of the Cas9-induced DSB (76). Using a DNA-free CRISPR-Cas9 approach avoids unwanted incorporation of the plasmid DNA into the cell's genome. DNA-free gene editing workflows can be uniquely and easily attained with synthetic gRNAs. Because unmodified RNAs are rapidly degraded in cells, gRNAs only need to be sufficiently stable to interact with Cas9, bind the target DNA, and allow cutting by Cas9 nuclease. In order to achieve rapid gene editing with synthetic gRNAs, the Cas9 nuclease needs to be readily available for interaction with the gRNA; this can be achieved when Cas9 is co-delivered as mRNA or protein and does not require transcription and/or translation, respectively.
Another advantage of the DNA-free system is to remove the variability that can arise from the choice of promoter used to drive expression from vector-based CRISPR-Cas9 systems. It is well known that not all promoters are functional in every cell type or cell line, so delivery of Cas9 protein or Cas9 mRNA avoids incompatibilities of certain promoters in specific cells. Overall, the functionality of the Cas9 and gRNA RNP complexes has been anecdotally reported as being superior to other methods of delivery, although a comprehensive comparison has not yet been performed.
6. CONCLUDING REMARKS AND FUTURE PERSPECTIVES
The use of synthetic gRNAs for CRISPR-Cas9 genome engineering has a number of advantages over vector-based components, including removing the time and labor for cloning sgRNA expression cassettes or generation and purification of in vitro transcribed sgRNAs, removing the potential for additional offtarget effects by prolonged sgRNA expression or avoiding unwanted genetic scarring through incorporation of vector sequence into the genome. Using synthetic oligonucleotide CRISPR-Cas9 components that can be added exogenously will enable better delivery and formulation strategies in cell culture as well as in other organisms (77). Chemically modified, conjugated, and/or nanoparticle formulated-gRNAs will enhance the stability of gRNAs that is required for some applications, increase gene editing potency, and potentially further reduce off-target effects. Additionally, the synthetic approach enables arrayed gene knockout screening in a high-throughput manner as well as offering a completely DNA-free solution. Synthetic RNAs in combination with non-viral delivery methods such as electroporation may be among the first strategies used for ex vivo editing in a cell therapy setting. Future versions of gRNA constructs with defined size, modification, structure, and functionality will continue to improve the efficacy and specificity of the CRISPR-Cas9 system.
SUPPLEMENTARY DATA STATEMENT
CONFLICT OF INTEREST
All authors are employed by Dharmacon. Some of the materials used in this study are products sold by Dharmacon. This does not alter our adherence to all of the Elsevier Publishing Ethics.
ACKNOWLEDGMENTS
This work was funded by Dharmacon, part of GE Healthcare. We thank Angela Schoolmeesters, Megan Basila and Eldon Chou for performing experiments and Louise Baskin for critical reading of the manuscript.
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FIGURE LEGENDS
Figure 1. The S. pyogenes CRISPR-Cas9 can be programmed by two separate RNAs or a long single guide RNA for efficient gene editing. (a) The native CRISPR-Cas9 system contains three components: the Cas9 protein (purple), the crRNA (green), and the tracrRNA (blue) which form a complex that binds to the target genomic DNA (gDNA, black) upstream of a protospacer-adjacent motif (PAM, red) and results in site-specific cleavage. (b) The sgRNA combines the crRNA and tracrRNA into one chimeric transcript for utility in generation of expression vectors and in vitro transcribed constructs. (c) Dual synthetic crRNA:tracrRNAs have similar gene editing efficiencies to synthetic sgRNAs or in vitro transcribed sgRNAs. HeLa and U2OS cells were plated at 10,000 cells/well one day prior to transfection. Cells were transfected with either Edit-R crRNA:tracrRNA (25 nM) or synthetic 99-mer sgRNA (25 nM) or in vitro transcribed 99-mer sgRNA (25 nM) and Edit-R Cas9 Nuclease protein (25 nM) using DharmaFECT Duo transfection reagent (0.4 pL/well) in biological triplicates (reagents available or synthesized at Dharmacon, part of GE Healthcare). A DNA mismatch detection assay using T7 Endonuclease I (T7EI; NEB) was performed 72 hours post-transfection to estimate gene editing efficiency (percentage under
Figure 2. Synthetic gRNAs can be applied to a variety of CRISPR-Cas9 experimental approaches. (a) Dual RNA can be co-delivered with Cas9 mRNA, Cas9 protein, or a Cas9 expression plasmid, or delivered into a stable Cas9-expressing cell line. Delivery can be achieved using a variety of methods resulting in efficient gene editing. (b) U2OS cells were plated at 10,000 cells/well one day prior to transfection. Cells were transfected with either Edit-R Cas9 Nuclease plasmid (200 ng), Edit-R Cas9 Nuclease mRNA (200 ng) or Cas9 nuclease protein (25 nM) and crRNA:tracrRNA (25 nM) targeting PPIB using DharmaFECT Duo transfection reagent (0.4 ^L/well) in biological triplicates. (c) U2OS-CAG-Cas9 stable cells were plated at 10,000 cells/well one day prior to transfection. Cells were transfected with crRNA:tracrRNA (25 nM) targeting PPIB using DharmaFECT 1 transfection reagent (0.2 ^L/well) in biological triplicates. A DNA mismatch detection assay using T7EI was performed 72 hours post-transfection to estimate gene editing efficiency (percentage under gel).
20 ±1 31 ±2 24 ±1
Figure 3. Synthetic gRNAs permit chemical modifications. Chemical modifications can be applied to the RNA backbone, base, and/or sugar. Covalent conjugations on 5' and 3' ends are also possible.
Figure 4. Chemical modifications enhance the nuclease stability of synthetic gRNAs for delivery by electroporation. (a) Effective gene editing requires sequential electroporation when using Cas9 mRNA and unmodified synthetic crRNA:tracrRNA. Effective gene editing with co-delivery of Cas9 mRNA and synthetic crRNA:tracrRNA requires the addition of modifications to the crRNA and tracrRNA molecules. crRNA and tracrRNA with three MS modifications on both the 5' and 3' ends (3xMS) provide nuclease resistance and allow for co-delivery by electroporation for gene editing. Electroporation of Cas9 mRNA (5 pg) and synthetic crRNA:tracrRNA (5.36 pM) into K562 cells (2 million) was performed using a Nucleofector™ 2b (Lonza). Sequential electroporation was performed with Cas9 mRNA first and followed by electroporation of unmodified synthetic crRNA:tracrRNA 6 hours later. A DNA mismatch detection assay using T7EI was performed 72 hours post-transfection to estimate gene editing efficiency (percentage under gel). (b) Modifications are not necessary for lipid transfection of synthetic crRNA:tracrRNA with Cas9 mRNA as similar levels of gene editing were calculated for 3xMS modified and unmodified dual RNA. U2OS cells were plated at 10,000 cells/well one day prior to transfection. Cells were transfected with Cas9 Nuclease mRNA (200 ng) and crRNA:tracrRNA (25 nM) using DharmaFECT Duo transfection reagent (0.4 pL/well) in biological triplicates. A DNA mismatch detection assay using T7EI was performed 72 hours post-transfection to estimate gene editing efficiency (percentage under
Sequential electroporation Co-electroporation
bp UT NTC unmodified _3xMS unmodified
bp 800
49 ±2 47 53 40
Lipid transfection UT NTC unmodified 3xMS
38 ± 1 39 ± 2
Figure 5. High throughput arrayed screening applications with phenotypic assays using dual RNAs. (a) A general workflow for screening with arrayed synthetic crRNA:tracrRNA, starting with generation of stable cells expressing Cas9 nuclease and ending with the phenotypic readout. U2OS-Ubi[G76V]-EGFP cells that stably express Cas9 under CAG promoter were generated and examined for editing efficiency using control crRNA (see Fig 2B). These U2OS-Ubi[G76V]-EGFP-CAG-Cas9 cells were then used in several downstream phenotypic assays (b-d). (b) Proteasome function assays: cells were seeded at 4,300 cells/well in black 96-well tissue culture plates and were transfected the following day with synthetic crRNA:tracrRNA (50 nM) targeting PPIB, PSMD7 or VCP genes using DharmaFECT 4 transfection reagent (0.07 ^g/well). After 72 hours the EGFP fluorescence was measured using an Envision plate reader (Perkin Elmer). (c) Apoptosis assay: cells seeded at 10,000 cells/well in 96-well format were transfected the following day with five different crRNA:tracrRNA (25 nM) targeting 16 different genes. PLK1 siRNA was used as a positive control (red) and non-targeting siRNA (NTC; green) and crRNA:tracrRNA targeting PPIB were used as negative controls. The effects on apoptosis were assayed using a Caspase-3/7 homogeneous assay (ApoONE, Promega) at 48 hours post-transfection. Data were normalized to negative control. (d) Mitotic Index: cells seeded at 5,000 cells/well in 96-well format were transfected the following day with crRNA:tracrRNA (25 nM) targeting PLK1 and KIF11 or non-targeting crRNA controls (NTC). Cells were fixed at 48 hours post-transfection and stained with anti-PH3 antibody and Hoechst 33342 before analysis on the IN Cell Analyzer 2200 imaging system (GE Healthcare).
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