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The CRISPR-Cas9 system has transformed genome engineering of model organisms from possible to practical. CRISPR-Cas9 can be readily programmed to generate sequence-specific double-strand breaks that disrupt targeted loci when repaired by error-prone non-homologous end joining or to catalyze precise genome modification through homology-directed repair (HDR). Here we describe a streamlined approach for rapid and highly efficient engineering of the Drosophila genome via CRISPR-Cas9-mediated HDR. In this approach, transgenic flies expressing Cas9 are injected with plasmids to express guide RNAs (gRNAs) and positively marked donor templates. We detail target site selection; gRNA plasmid generation; donor template design and construction; and the generation, identification and molecular confirmation of engineered lines. We also present alternative approaches and highlight key considerations for experimental design. The approach outlined here can be used to rapidly and reliably generate a variety of engineered modifications, including genomic deletions and replacements, precise sequence edits, and incorporation of protein tags.
The CRISPR-Cas9 system is significantly advancing the ability of researchers to engineer targeted genome modifications for functional studies of genes and genetic elements. In Drosophila, the CRISPR-Cas9 system has been used to disrupt, delete, replace, tag and edit multiple genes and genetic elements (Bassett et al., 2013; Gratz et al., 2013a; Gratz et al., 2014; Kondo and Ueda, 2013; Lee et al., 2014; Port et al., 2014; Ren et al., 2013; Sebo et al., 2014; Xue et al., 2014; Yu et al., 2014; Yu et al., 2013). The rapid and widespread adoption of CRISPR-Cas9 illustrates the utility of this novel genome engineering platform for generating a wide variety of modifications, and its power for addressing fundamental biological questions, understanding and treating disease, and engineering agriculturally relevant species and their pests.
Endogenous CRISPR-Cas9 systems have been adapted as simple and highly robust genome engineering tools that are being widely adopted by the research community. The most widely used Streptococcus pyogenes system was simplified to two components to facilitate genome engineering: a common endonuclease called Cas9 and a single chimeric RNA referred to as a guide RNA (gRNA) (Jinek et al., 2012). gRNAs interact with Cas9 and guide the nuclease to specific DNA sequences through an easily programmed 20-nt target sequence that directly base pairs with complementary DNA. Upon binding its target, Cas9 utilizes its two nuclease domains to generate a double-strand break (DSB). The only known requirement for a potential cleavage site is the presence of a 3-bp protospacer adjacent motif (PAM) of the form NGG immediately 3′ of the 20-nt target sequence. Thus, S. pyogenes CRISPR-Cas9 target sites occur an average of once in every eight basepairs of genomic sequence.
Induction of a DSB in genomic DNA triggers repair by one of two general cellular repair pathways, both of which can be co-opted for genome engineering. Non-homologous end-joining (NHEJ) is an error-prone process in which broken ends are simply ligated together. This repair pathway can yield small insertions and deletions (indels) that disrupt function at cleavage sites. In contrast, homology-directed repair (HDR) employs homologous DNA sequences as templates for precise repair. By supplying donor templates comprising exogenous sequence flanked by homology-containing stretches (commonly referred to as homology arms), the HDR pathway can be appropriated to make precise modifications including defined deletions, sequence substitutions, or insertions. Beyond the genome engineering applications of the CRISPR-Cas9 system, nuclease-dead Cas9 is being used as a sequence-specific repressor or activator of gene expression and is being developed as a tool for probing genome structure and function without causing mutations (Anton et al., 2014; Bikard et al., 2013; Chen et al., 2013; Cheng et al., 2013; Fujita and Fujii, 2013; Gilbert et al., 2013; Kearns et al., 2014; Maeder et al., 2013; Perez-Pinera et al., 2013; Qi et al., 2013)
Here we detail a rapid and efficient CRISPR-Cas9 method for HDR-mediated engineering of the Drosophila genome (Gratz et al., 2013a; Gratz et al., 2014). We have used this approach to generate numerous genome modifications, including gene replacements, in-frame protein tag insertions, and conditional alleles. The Basic Protocol covers target site selection; gRNA generation; donor design and construction; and the generation, identification and molecular confirmation of engineered lines. We begin with key considerations for experimental design and discuss alternative approaches.
Figure 1 shows a decision tree that can be used as a guide in designing the appropriate strategy for different types of CRISPR-Cas9 genome engineering experiments. Here we discuss the key considerations for each decision point.
In the Basic Protocol below, we detail our preferred method for efficient generation of engineered flies via HDR: injection of vasa-Cas9 flies with gRNA plasmids and a positively marked dsDNA donor template. This choice represents a favorable balance of time, cost, efficiency, and reliability. With this approach, we obtain engineered alleles within one month at a total reagent cost of approximately $150. Injections generally cost an additional $200 if outsourced. In our experience, an average of 25% (range = 7–42%) of fertile injected flies transmit the targeted event to their progeny. In Alternate Protocol 1, we detail HDR with ssDNA donor templates. Alternate Protocol 2 covers HDR in other genetic backgrounds using Cas9 supplied as DNA. In Alternate Protocol 3, we outline our approach for NHEJ using a transgenic source of Cas9 and gRNA supplied as DNA. This approach has also been used successfully by Ren et al. (2013). Together these protocols offer a versatile toolset amenable for generating a variety of genome modifications in Drosophila.
Selection of high-quality target sites is essential for the success of any CRISPR-based genome engineering experiment. It is important to identify target sites that will generate DSBs close to the location of the intended modification. In choosing a target site, location must be balanced with target-site specificity and, thus, the potential for off-target DSBs. While originally raised as a significant concern in the editing of transformed cell lines (Fu et al., 2013), with careful target site selection, off-target cleavage does not seem to be a significant problem for genome editing of organisms or human stem cells (Bassett et al., 2013; Chiu et al., 2013; Duan et al., 2014; Gratz et al., 2013a; Gratz et al., 2014; Kiskinis et al., 2014; Smith et al., 2014; Suzuki et al., 2014; Veres et al., 2014; Yang et al., 2013). Nonetheless, our current understanding of Cas9-induced cleavage is far from complete, so it is important to select the most specific sites possible to minimize the potential for off-target mutagenesis. To facilitate the rapid identification of high-quality target sites, we have developed a web-based tool, CRISPR Optimal Target Finder, that identifies gRNA cleavage sites and evaluates their specificity (Gratz et al., 2014).
It is essential that target sites be identified in sequence obtained from the fly strain that will be edited, not the reference genome. Polymorphisms between a given fly strain and the reference genome are frequent, especially in intergenic regions, and could eliminate or significantly decrease cleavage if they occur within your target sequence. Thus, CRISPR Optimal Target Finder identifies gRNA target sites in user-supplied DNA sequence rather than reference genome sequences. In the Basic Protocol, we use vasa-Cas9 flies. However, as described in Alternate Protocol 2 below, our approach can be readily adapted to engineer any fly strain.
To supply gRNAs containing the target-specific sequences from a plasmid DNA source, we have generated vectors for rapid cloning of target-specific sequences using short complementary oligonucleotides and a simple annealing and ligation process. The pU6-BbsI-gRNA vectors utilize the small RNA promoter of a Drosophila U6 gene to express the gRNA.
dsDNA donor vectors can be made in many configurations to facilitate the generation of an endless variety of genome modifications (Figure 3). The Basic Protocol focuses on the design of donor constructs using the pHD-DsRed-attP or pHD-DsRed vectors available through Addgene. The design of ssDNA donors is described in Alternate Protocol 1 below. The pHD-DsRed-attP vector is used for generating marked knock-out alleles that harbor an attP phage recombination site for serial manipulations of the target locus catalyzed by phiC31. pHD-DsRed is used for generating positively marked targeted insertions or sequence edits. Both vectors include a removable DsRed marker expressed strongly in the eye for visual identification of lines with targeted events and contain multiple cloning sites for inserting locus-specific homology arms.
Below we outline the steps for rapidly constructing dsDNA donor constructs using the type IIS restriction sites AarI and SapI in the pHD-DsRed-attP or pHD-DsRed vectors for seamless integration of homology arms. These vectors also contain multiple cloning sites for an alternative cloning method.
|1 cycle:||2 min||94°C||(initial denaturation)|
|30 cycles:||10 sec||98°C||(denaturation)|
|1 cycle:||10 min||72°C||(final extension)|
CRISPR components are injected using standard Drosophila injection techniques. Here we provide the injection mixture for HDR in vasa-Cas9 flies.
Following injection and an appropriate outcross of injected flies, candidate CRISPR alleles are easily identified by screening for flies with red fluorescent eyes in F1 progeny. Once these candidates have been crossed to a balancer line, they can be sacrificed for molecular characterization to verify recovery of the intended genome modification. Below we describe our strategy of performing three PCRs that, in combination with Sanger sequencing, confirm targeted and precise editing (See Figure 3).
When engineering small modifications, it may be desirable to use ssDNA donors, which can be rapidly synthesized. However, ssDNA donors are generally limited to 200 nt and, thus, cannot be used for engineering large modifications (such as the integration of a fluorescent tag). They also cannot be designed to include a visible marker for screening, so molecular screening is required, which increases the time and labor required to recover engineered flies.
For many applications, it is necessary or desirable to engineer a specific fly strain. This is easily accomplished using an injectable source of Cas9 such as pBS-Hsp70-Cas9. Targeting efficiency is lower than with a transgenic Cas9 source, so it is advisable to inject a larger number of embryos.
If your goal is to generate a disruptive allele, you can target the NHEJ repair pathway by introducing Cas9 and one or two gRNAs in the absence of a donor repair template. Using one gRNA to target a single cleavage event in critical sequence, you can recover disruptive indels. With two gRNAs, you can delete the intervening sequence.
Adult fly homogenization buffer:
The CRISPR-Cas9 system is a highly accessible and effective tool for genome engineering in Drosophila (Bassett and Liu, 2014; Gratz et al., 2013b; Harrison et al., 2014; Kondo, 2014). The Basic Protocol outlined above details an optimized CRISPR-Cas9 approach that has several advantages. We use a transgenic source of Cas9, expressed in the germline under the control of the vasa promoter, to achieve highly efficient and reliable genome engineering. The introduction of gRNA using rapidly constructed plasmids is quick and inexpensive. Our donor vectors facilitate streamlined cloning of locus-specific donor templates, and the incorporation of a removable DsRed marker makes identification of candidate alleles markedly easier than identification through molecular characterization.
While not covered in our protocol, other groups have successfully applied the CRISPR-Cas9 system in Drosophila using a variety of methods for introducing gRNAs and Cas9. NHEJ has been successfully accomplished using transgenic Cas9 + gRNA supplied as RNA (Xue et al., 2014), transgenic Cas9 + transgenic gRNA (Kondo and Ueda, 2013; Port et al., 2014; Xue et al., 2014), Cas9 DNA + gRNA plasmid (Gratz et al., 2013a; Ren et al., 2013), and Cas9 mRNA + gRNA supplied as RNA (Bassett et al., 2013; Yu et al., 2013). Successful HDR has been reported using Cas9 DNA, gRNA plasmid and either a dsDNA or ssDNA donor (Gratz et al., 2013a; Gratz et al., 2014). However, efficiency is higher with a transgenic Cas9 source, and all other HDR experiments reported in Drosophila to date have been conducted in Cas9-expressing flies using either gRNA plasmid (Gratz et al., 2014), gRNA supplied as RNA (Xue et al., 2014; Yu et al., 2014), or a transgenic gRNA source (Port et al., 2014).
Sequencing of target sites: Due to the prevalence of polymorphisms between distinct genetic backgrounds in Drosophila, it is critical to sequence the intended target locus in the genetic background in which the genome engineering experiment will be performed. Even a single basepair change in a target site can be detrimental to the success of the experiment.
Donor template construction: To protect both the donor template and the modified locus from unintended cleavage, it is critical that your donor template not contain an intact gRNA target site
Molecular confirmation of engineered lines: Because unexpected events can always occur during DNA repair, it is important to thoroughly confirm all candidate alleles. To do this, we suggest three PCRs (See Figure 3B) that together will confirm that engineered DNA has been incorporated at the target locus and that the locus is free of additional modifications, including the integration of donor vector backbone sequences (Yu et al., 2014).
Finally, it is important to note that, while CRISPR-Cas9 is working quite well in Drosophila, the system is not yet fully understood. For example, locus- and sequence-specific effects on cleavage efficiency are poorly understood. The Perrimon group (Harvard Medical School) has developed a tool that uses data from high throughput experiments in S2 cells to predict cleavage efficiency based on gRNA target sequence (www.flyrnai.org/evaluateCrispr/). An understanding of how donor composition and other experimental design features may influence the efficiency of HDR or the DNA repair pathway utilized by the cell awaits further study.
Poor viability: Reduce the concentration of gRNAs and donor vector in the injection mixture. While this may reduce efficiency, we have found that reducing the overall concentration of the injection mixture can increase viability. If the poor viability is due to highly efficient generation of a lethal allele, use an injected DNA source of Cas9 (pBS-hsp70-Cas9) instead of vasa-Cas9. This will decrease cleavage efficiency, and thus the occurrence of biallelic events, facilitating the recovery of recessive lethal lesions.
Poor efficiency: In the event that a given targeting experiment fails to yield engineered alleles, the gRNAs should be tested for cleavage efficiency. For genome engineering strategies using a pair of gRNAs, a simple PCR spanning the two gRNA target sites can be performed on embryos 24 hours after injection of both gRNAs into vasa-Cas9. Amplicons indicating a deletion between the two targeted cleavage sites demonstrate that both gRNAs are capable of generating DSBs. For strategies using one gRNA, cleavage efficiency can be tested using HRMA or a mismatch-specific nuclease assay on embryos 24 hours post injection. This approach can also be used to assess gRNA efficiency prior to embarking on a CRISPR-based experiment.
Using the approach described above to replace genes with attP docking sites in multiple experiments, an average of 24% of injected flies produced correctly engineered progeny. The deleted genes ranged in size from 2 to 27 Kb. Interestingly, we have not observed a strong correlation between deletion size and efficiency, suggesting that locus- or gRNA-specific effects may play a larger role in determining differences in efficiency between targeting experiments. We have also used this approach to insert in-frame tags in a number of loci at a similar average efficiency of 26%.
The highest probability off-target cleavage sites can be identified by sequence similarity to the targeted site. In a subset of our experiments, we have assayed these sites and found no evidence of off-target cleavage (Gratz et al., 2014). Based on this and similar findings by others, we expect that with careful target site selection, engineered fly lines can be generated with few or no off-target mutations in most cases (Bassett et al., 2013; Gratz et al., 2013a; Gratz et al., 2014).
Target site selection, preparation of gRNA plasmids, and construction of donor templates can be accomplished in 1 week with 1–4 hours hands-on time per day. Embryo injection can be accomplished in 1 day with 4 hours hands-on time. After 10 days, injected flies can be outcrossed in approximately 1 hour. After another 10 days, F1 progeny are screened and outcrossed (2 hours) before molecular confirmation, which can be completed in 2 days (5 hours hands-on time).
We thank the members of the Harrison, Wildonger and O’Connor-Giles labs for invaluable input throughout this work. Plasmids and transgenic fly lines described here are available through the non-profit distributor Addgene and the Bloomington Drosophila Stock Center, respectively. Additional information and resources are available at flyCRISPR.molbio.wisc.edu and tools.flycrispr.molbio.wisc.edu/targetFinder. All software code is available upon request. This work was funded by startup funds from the University of Wisconsin, a grant from the McKnight Foundation to KOCG, grants from the National Institute of Neurological Disorders and Stroke, National Institutes of Health to KOCG (R01 NS078179 and R21 NS088830) and JW (R00 NS072252), and grants from the Wisconsin Partnership Program and March of Dimes to M.M.H.
CRISPR fly design
Bloomington Drosophila Stock Center CRISPR stocks
Addgene CRISPR plasmids
Target finder tools:
CRISPR Optimal Target Finder
DRSC Find CRISPRs
flyCRISPR Discussion group