X4. CRISPR

information about CRISPR

http://www.nature.com/news/five-big-mysteries-about-crispr-s-origins-1.21294

Overview of CRISPR/Cas9

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Type II system is a bacterial immune system that has been modified for genome engineering (see CRISPR history). Prior to CRISPR/Cas9, genome engineering approaches, like zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALENs), relied upon the use of customizable DNA-binding protein nucleases that required scientists to design and generate a new nuclease-pair for every genomic target. Largely due to its simplicity and adaptability, CRISPR has rapidly become one of the most popular approaches for genome engineering.

CRISPR consists of two components: a “guide” RNA (gRNA) and a non-specific CRISPR-associated endonuclease (Cas9). The gRNA is a short synthetic RNA composed of a “scaffold” sequence necessary for Cas9-binding and a user-defined ∼20 nucleotide “spacer” or “targeting” sequence which defines the genomic target to be modified. Thus, one can change the genomic target of Cas9 by simply changing the targeting sequence present in the gRNA. CRISPR was originally employed to “knock-out” target genes in various cell types and organisms, but modifications to the Cas9 enzyme have extended the application of CRISPR to selectively activate or repress target genes, purify specific regions of DNA, and even image DNA in live cells using fluorescence microscopy. Furthermore, the ease of generating gRNAs makes CRISPR one of the most scalable genome editing technologies and has been recently utilized for genome-wide screens.

This guide will provide a basic understanding of CRISPR/Cas9 biology, introduce the various applications of CRISPR, and help you get started using CRISPR/Cas9 in your own research.

Generating a Knock-out Using CRISPR/Cas9 

CRISPR/Cas9 can be used to generate knock-out cells or animals by co-expressing a gRNA specific to the gene to be targeted and the endonuclease Cas9. The genomic target can be any ∼20 nucleotide DNA sequence, provided it meets two conditions:

• 1. The sequence is unique compared to the rest of the genome.

  2. The target is present immediately upstream of a Protospacer Adjacent Motif (PAM).

The PAM sequence is absolutely necessary for target binding and the exact sequence is dependent upon the species of Cas9 (5′ NGG 3′ for Streptococcus pyogenes Cas9). We will focus on Cas9 from S. pyogenes as it is currently the most widely used in genome engineering (see additional species of Cas9 and corresponding PAM sequences here). Once expressed, the Cas9 protein and the gRNA form a riboprotein complex through interactions between the gRNA “scaffold” domain and surface-exposed positively-charged grooves on Cas9. Cas9 undergoes a conformational change upon gRNA binding that shifts the molecule from an inactive, non-DNA binding conformation, into an active DNA-binding conformation. Importantly, the “spacer” sequence of the gRNA remains free to interact with target DNA. The Cas9-gRNA complex will bind any genomic sequence with a PAM, but the extent to which the gRNA spacer matches the target DNA determines whether Cas9 will cut. Once the Cas9-gRNA complex binds a putative DNA target, a “seed” sequence at the 3′ end of the gRNA targeting sequence begins to anneal to the target DNA. If the seed and target DNA sequences match, the gRNA will continue to anneal to the target DNA in a 3′ to 5′ direction.

Cas9 will only cleave the target if sufficient homology exists between the gRNA spacer and target sequences. The “zipper-like” annealing mechanics may explain why mismatches between the target sequence in the 3′ seed sequence completely abolish target cleavage, whereas mismatches toward the 5′ end are permissive for target cleavage. The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Cas9 undergoes a second conformational change upon target binding that positions the nuclease domains to cleave opposite strands of the target DNA. The end result of Cas9-mediated DNA cleavage is a double strand break (DSB) within the target DNA (∼3-4 nucleotides upstream of the PAM sequence).

The resulting DSB is then repaired by one of two general repair pathways:

• 1. The efficient but error-prone Non-Homologous End Joining (NHEJ) pathway

  2. The less efficient but high-fidelity Homology Directed Repair (HDR) pathway

The NHEJ repair pathway is the most active repair mechanism, capable of rapidly repairing DSBs, but frequently results in small nucleotide insertions or deletions (InDels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical implications, because a population of cells expressing Cas9 and a gRNA will result in a diverse array of mutations (for more information, jump to Plan Your Experiment). In most cases, NHEJ gives rise to small InDels in the target DNA which result in in-frame amino acid deletions, insertions, or frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene. Ideally, the end result is a loss-of-function mutation within the targeted gene; however, the “strength” of the knock-out phenotype for a given mutant cell is ultimately determined by the amount of residual gene function.

Browse Plasmids: Double Strand Break (Cut)

Enhancing Specificity with Cas9 Nickase

CRISPR/Cas9 is highly specific when gRNAs are designed correctly, but specificity is still a major concern, particularly as CRISPR is being developed for clinical use. The specificity of the CRISPR system is determined in large part by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome. Ideally, a gRNA targeting sequence will have perfect homology to the target DNA with no homology elsewhere in the genome. Realistically, a given gRNA targeting sequence will have additional sites throughout the genome where partial homology exists. These sites are called “off-targets” and need to be considered when designing a gRNA for your experiment (more information on gRNA design can be found in the below Plan Your Experiment section).

In addition to optimizing gRNA design, specificity of the CRISPR system can also be increased through modifications to Cas9 itself. As discussed previously, Cas9 generates double strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. The exact amino acid residues within each nuclease domain that are critical for endonuclease activity are known (D10A for HNH and H840A for RuvC in S. pyogenesCas9) and modified versions of the Cas9 enzyme containing only one active catalytic domain (called “Cas9 nickase”) have been generated. Cas9 nickases still bind DNA based on gRNA specificity, but nickases are only capable of cutting one of the DNA strands, resulting in a “nick”, or single strand break, instead of a DSB. DNA nicks are rapidly repaired by HDR (homology directed repair) using the intact complementary DNA strand as the template (jump to our HDR section for more details). Thus, two nickases targeting opposite strands are required to generate a DSB within the target DNA (often referred to as a “double nick” or “dual nickase” CRISPR system). This requirement dramatically increases target specificity, since it is unlikely that two off-target nicks will be generated within close enough proximity to cause a DSB. Therefore, if specificity and reduced off-target effects are crucial, consider using the dual nickase approach to create a double nick-induced DSB. The nickase system can also be combined with HDR-mediated gene editing for highly specific gene edits.

Browse Plasmids: Single Strand Break (Nick)

Making Precise Modifications Using Homology Directed Repair (HDR)

While NHEJ-mediated DSB repair is imperfect and often results in disruption of the open reading frame of the gene, Homology Directed Repair (HDR) can be used to generate specific nucleotide changes (also known as gene “edits”) ranging from a single nucleotide change to large insertions (e.g. addition of a fluorophore or tag). 

In order to utilize HDR for gene editing, a DNA “repair template” containing the desired sequence must be delivered into the cell type of interest with the gRNA(s) and Cas9 or Cas9 nickase. The repair template must contain the desired edit as well as additional homologous sequence immediately upstream and downstream of the target (termed left & right homology arms). The length and binding position of each homology arm is dependent on the size of the change being introduced. The repair template can be a single stranded oligonucleotide, double-stranded oligonucleotide, or double-stranded DNA plasmid depending on the specific application. It is worth noting that the repair template must lack the PAM sequence that is present in the genomic DNA, otherwise the repair template becomes a suitable target for Cas9 cleavage. For example, the PAM could be mutated such that it is no longer present, but the coding region of the gene is not affected (i.e. a silent mutation).

The efficiency of HDR is generally low (<10% of modified alleles) even in cells that express Cas9, gRNA and an exogenous repair template. For this reason, many laboratories are attempting to artificially enhance HDR by synchronizing the cells within the cell cycle stage when HDR is most active, or by chemically or genetically inhibiting genes involved in NHEJ. The low efficiency of HDR has several important practical implications. First, since the efficiency of Cas9 cleavage is relatively high and the efficiency of HDR is relatively low, a portion of the Cas9-induced DSBs will be repaired via NHEJ. In other words, the resulting population of cells will contain some combination of wild-type alleles, NHEJ-repaired alleles, and/or the desired HDR-edited allele. Therefore, it is important to confirm the presence of the desired edit experimentally, and if necessary, isolate clones containing the desired edit (see our validation section in Plan Your Experiment).

Activation or Repression of Target Genes Using CRISPR/Cas9

The CRISPR/Cas system is a remarkably flexible tool for genome manipulation. A unique feature of Cas9 is its ability to bind target DNA independently of its ability to cleave target DNA. Specifically, both RuvC- and HNH- nuclease domains can be rendered inactive by point mutations (D10A and H840A in SpCas9), resulting in a nuclease dead Cas9 (dCas9) molecule that cannot cleave target DNA. The dCas9 molecule retains the ability to bind to target DNA based on the gRNA targeting sequence. The first experiments using dCas9 in bacteria demonstrated that targeting dCas9 to transcriptional start sites was sufficient to “repress” or “knock-down” transcription by blocking transcription initiation. Furthermore, dCas9 can be tagged with transcriptional repressors or activators, and targeting these dCas9 fusion proteins to the promoter region results in robust transcription repression or activation of downstream target genes. The simplest dCas9-based activators and repressors consist of dCas9 fused directly to a single transcriptional activator, A (e.g. VP64) or transcriptional repressors, R (e.g. KRAB; see A in Figure to the right). Additionally, more elaborate activation strategies have been developed which result in greater activation of target genes in mammalian cells. These include: co-expression of epitope-tagged dCas9 and antibody-activator effector proteins (e.g. SunTag system, B), dCas9 fused to several different activation domains in series (e.g. dCas9-VPR, C) or co-expression of dCas9-VP64 with a “modified scaffold” gRNA and additional RNA-binding “helper activators” (e.g. SAM activators, D). Importantly, unlike the genome modifications induced by Cas9 or Cas9 nickase, dCas9-mediated gene activation or repression is reversible, since it does not permanently modify the genomic DNA.

Browse Plasmids: Activate, Repress/Interfere

Multiplex Genome Engineering with CRISPR/Cas9

Expressing several gRNAs off of the same plasmid ensures that every cell that takes up a plasmid expresses all of the desired gRNAs and increases the likelihood that all desired genomic edits will be carried out by Cas9. Such “multiplex” CRISPR applications include:

• • The use of dual nickases to generate a knock-out or edit a gene

  • Using Cas9 to generate large genomic deletions

  • Modifying multiple different genes at once

Current multiplex CRISPR systems enable researchers to target anywhere from 2 to 7 genetic loci by cloning multiple gRNAs into a single plasmid. These multiplex gRNA vectors can conceivably be combined with any of the aforementioned Cas9-derivatives to not only knock-out target genes, but activate or repress target genes as well.

Browse Plasmids: Multiplex gRNA vectors

Genome-wide Screens Using CRISPR/Cas9

The ability to semi-automatically design and synthesize gRNAs to mutate, activate, or repress almost any genomic locus makes the CRISPR/Cas9 the ideal genome engineering system for large-scale forward genetic screening. Forward genetic screens are particularly useful for studying diseases or phenotypes for which the underlying genetic cause is not known. In general, the goal of a genetic screen is to generate a large population of cells with mutations in a wide variety of genes and use these mutant cells to identify the genetic perturbations that result in a desired phenotype. Before CRISPR/Cas9, genetic screens relied heavily on shRNA-based screens, which are prone to off-target effects and may result in false negatives due to incomplete knock-down of target genes. The CRISPR system, in contrast, is capable of making highly specific, permanent genetic modifications in target genes. The CRISPR system has already been used to screen for novel genes that regulate known phenotypes, including resistance to chemotherapy drugs, resistance to toxins, cell viability, and tumor metastasis. Currently, the most popular method for conducting genome-wide screens using CRISPR/Cas9 involves the use of “pooled” lentiviral CRISPR libraries.

What are pooled lentiviral CRISPR libraries?

Pooled lentiviral CRISPR libraries (heretofore referred to as CRISPR libraries) are a heterogenous population of lentiviral transfer vectors, each containing an individual gRNA targeting a single gene in a given genome.

Guide RNAs are designed in silico and synthesized (see A in figure to the right), then cloned in a pooled format into lentiviral transfer vectors B. CRISPR libraries have been designed for most of the common CRISPR applications including genetic knock-out and activation or repression for both human and mouse genes. Although each library is different, there are several features that are common across most CRISPR libraries. Each library typically contains ∼3-6 gRNAs per gene to ensure modification of every target gene. Libraries can target anywhere from a single class of genes up to every gene in the genome. Thus, CRISPR libraries contain thousands of unique gRNAs targeting a wide variety of genes. Guide RNA design for CRISPR libraries follows the same general principles as designing a gRNA for a specific target. Target sequences must be unique compared to the rest of the genome and be located just upstream of a PAM sequence. Obviously, the exact region of the gene to be targeted may vary depending on the specific application (5′ constitutively expressed exons for knock-out libraries, or the promoter region for activation and repression libraries). For some libraries, Cas9 (or Cas9 derivative) is included on the gRNA-containing plasmid; for others, they must be delivered to the cells separately.

How does one use a CRISPR library?

All of the CRISPR libraries available through Addgene follow the same general experimental protocol. In most cases, the CRISPR library will be shipped at a concentration that is too low to be used in experiments. Thus, the first step in using your library is to “amplify” the library (C in above figure) such that the total amount of DNA is increased but the “representation” (i.e. the relative percentage of each gRNA with the library) is maintained. Once the library has been amplified and the representation checked using next-generation sequencing (NGS), the next step is to generate lentivirus containing the entire CRISPR library D. Mutant cells are then generated by transducing Cas9-expressing cells (or wild-type cells for libraries containing Cas9 and the gRNA) with the lentiviral library E. In screens where you are measuring the loss of gRNAs from a final population (i.e. negative selection survival screens) you need to use NGS to identify the gRNAs present in the initial mutant cell population prior to selection. Alternatively, for positive screens such as drug-screens, you can treat your mutant cells with drug, or control and directly compare the gRNA distribution at the end of the screen F. It is important to remember that analysis of relevant genes (“hits”) at the end of your screen requires the use of NGS.

What can screens tell you?

As noted at the beginning of this section, forward genetic screens are most useful for situations in which the physiology or cell biology behind a particular phenotype or disease is well understood, but the underlying genetic causes are unknown. Therefore, genome-wide screens using CRISPR libraries are a great way to gather unbiased information regarding which genes, if any, play a causal role in a given phenotype. With any experiment, it is important to be sure that the hits you identify are actually important for your phenotype. This is typically carried out by testing the gRNAs identified in your screen individually to ensure that the genetic modification reproduces the phenotype you screened for in the first place.

Browse Libraries: CRISPR Pooled Libraries

Additional Uses of Cas9

Image Genomic Regions Using Fluorophore-tagged dCas9

Using a dCas9 fused to a fluorescent marker (such as GFP), researchers have turned dCas9 into a customizable DNA label that can be detected in live cells. By creating unique gRNAs that bind in close proximity along a stretch of genomic DNA, a technique referred to as “tiling”, researchers have imaged specific regions of the genome. The tiling technique does require multiple gRNAs to bind near one another in order to produce a detectable signal.

Browse Plasmids: Label

Purify Genomic Regions Using dCas9

Building on the well-established concept of ChIP (Chromatin Immunoprecipitation), researchers have created enChIP (engineered DNA-binding molecule-mediated ChIP) that allows for the purification of any genomic sequence specified by a particular gRNA. A catalytically inactive dCas9 fused to an epitope tag(s) can be used to purify genomic DNA bound by the gRNA.  Learn more about ChIP here.

Browse Plasmids: Purify

Alternatives to Cas9 for CRISPR Genome Engineering

While S. pyogenes Cas9 (SpCas9) is certainly the most commonly used CRISPR endonuclease for genome engineering, it may not be the best endonuclease for every application. For example, the PAM sequence for SpCas9 (5′ NGG 3′) is abundant throughout the human genome, but a NGG sequence may not be positioned correctly to target your desired genes for modification. This is of particular concern when trying to edit a gene using Homology Directed Repair (HDR), which requires PAM sequences in very close proximity to the region to be edited. In the Joung lab, Kleinsteiver et al. has generated synthetic SpCas9-derived variants with non-NGG PAM sequences. The inclusion of these variants into the CRISPR arsenal effectively doubles the targeting range of CRISPR in the human genome.

Additional Cas9 orthologs from various species have been identified and these “non-SpCas9s” bind a variety of PAM sequences. These non-SpCas9s may have other characteristics that make them more useful than SpCas9 for specific applications. For example, the relatively large size of SpCas9 (∼4kb coding sequence) means that plasmids carrying the SpCas9 cDNA cannot be efficiently packaged into adeno-associated virus (AAV). Conversely, the coding sequence for Staphylococcus aureus Cas9 (SaCas9) is ∼1 kilobase shorter than SpCas9, allowing it to be efficiently packaged into AAV. Similar to SpCas9, the SaCas9 endonuclease is capable of modifying target genes in mammalian cells in vitro and in mice in vivo.

Another limitation of SpCas9 has to do with the low efficiency of making specific genetic edits via HDR. In the Zhang lab, Zetsche et al. describes two RNA-guided endonucleases from the Cpf1 family that display cleavage activity in mammalian cells. Unlike Cas9 nucleases, the result of Cpf1-mediated DNA cleavage is a double-strand break with a short 3′ overhang. Cpf1’s staggered cleavage pattern opens up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Like the Cas9 variants and orthologs described above, Cpf1 also expands the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM sites favored by SpCas9.