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With so many options available, it can be difficult to choose a gene silencing method. This article will shed some light on the differences between CRISPR, RNAi, and TALEN, and when it would be beneficial to use each method.

CRISPR and TALEN are similar in that they cause gene knockout, whereas RNAi causes gene knockdown. What’s the difference?

Gene knockout can occur when a double stranded break has been made to the DNA within the coding region of a gene. The event triggers one of two repair pathways: either Non-Homologous End Joining (NHEJ) or Homology Directed Repair (HDR) (if a repair template is provided). Because NHEJ is an error-prone process, it will often result in the insertion or deletion of nucleotides (called InDel mutations). Because each cell will undergo a different editing event, screening is necessary to isolate a cell line that has frameshift mutations resulting in a significant change in the translated protein (often truncation). If a cell line has a frameshift mutation in all alleles of a gene, the protein will be completely knocked out. This means that there will be no expression of functional protein in the cell.

Gene knockdown refers to when expression of a gene is reduced, but not completely silenced. This is commonly accomplished by targeting the mRNA transcript of a gene instead of the DNA. The mRNA is either degraded or translation is blocked. This means that some gene expression usually escapes regulation.

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a. CRISPR

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. This new gene editing technology is powerful due to its simple approach to design and its flexibility.

The engineered CRISPR system is composed of two parts: a single guide RNA (sgRNA) and Cas9 nuclease. These components come together to form a ribonucleoprotein (RNP). The ~20 bp at the 5’ end of the sgRNA (the crRNA) guides the Cas9 nuclease to a specific target sequence in the genome. In order for Cas9 to cleave the DNA, it must first recognize a DNA sequence Protospacer Adjacent Motif (PAM) sequence to the 3’ of the target sequence. The PAM sequence required by Cas9 differs depending on which species the Cas9 nuclease originates from; spCas9 from Streptococcus pyogenes has a PAM sequence of 5’-NGG, while saCas9 from S. aureus has a PAM sequence of 5’-NNGRRT.

After binding the DNA, Cas9 will cause a double stranded break at the target site. Error-prone NHEJ repair of the site by the cell will induce indel mutations. Because each cell will undergo a different editing event, screening is necessary to isolate a cell line that has frameshift mutations resulting in a significant change in the translated protein (often truncation), leading to gene knockout.

For a more comprehensive overview, please see our Introduction to CRISPR Knowledge Base article.

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b. TALEN

TALEN stands for Transcription Activator-Like Effector Nucleases. TALENs are artificial restriction enzymes that consist of a TAL effector DNA-binding domain fused to the DNA nuclease domain from the enzyme FokI. The TAL effector DNA-binding domain is composed of 33-35 amino acid repeats, which differ from each other by two amino acids (called the Repeat-Variable Di-residue (RVD)). The identity of those two amino acids determines which nucleotide each repeat will bind. A stretch of 12 to 31 repeats can be assembled in a row in order to target the TALEN to a specific DNA sequence in the genome.

In order for the nuclease domain to cut, the TALEN must dimerize. This means that two different TALENs must be supplied to the cell: one targeting each strand of DNA, separated by a small spacer sequence of 12-25 bp. After FokI cuts, it will cause a dsDNA break, which will be repaired by the cell’s error-prone NHEJ pathway. As with CRISPR, this will sometimes result in InDel frameshift mutations which can knockout gene expression.

c. RNAi

RNA interference (RNAi) exploits the endogenous system for miRNA-induced gene silencing to artificially inhibit gene expression via transcriptional regulation (for more information about miRNAs, see our Introduction to miRNA Knowledge Base article).

Short RNAs can be introduced to the cell as either shRNAs (short hairpin RNAs) or siRNAs (small interfering RNA). Both shRNAs and siRNAs are ~21 bp long and are designed to have complementarity to the target mRNA. shRNAs are dsRNAs that contain a loop structure, and are processed into siRNA by the host enzyme DICER. siRNA are dsRNA containing 2 nt 3’ end overhangs.

After processing, one strand of the siRNA will be loaded into the RISC (RNA-induced silencing complex). The siRNA will bind to its target based on complementarity. If the binding between the siRNA and the mRNA is perfect, the RISC will cleave the mRNA. If the binding between the siRNA and the mRNA is not perfect it will cause translational inhibition, but no mRNA cleavage will occur.

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In this section, we’ll compare each of the gene silencing methods using various criteria including ease of experimental design and use, number of off-target effects, cost, and time.

a. Ease of Design

Design of siRNAs is the easiest method, followed by design of sgRNAs for CRISPR and TALENs. In order to design siRNAs, one only needs the sequence of the corresponding mRNA transcript. Design for CRISPR and TALEN requires knowledge of the genomic DNA sequence.

siRNAs can be designed to target any transcript at almost any locus, although some sites are predicted to be more effective based on factors such as distance from transcription start site, nucleotide composition, and secondary structures in the target site (1).

In comparison, CRISPR depends on the presence of a PAM sequence in the gene of interest. Depending on the type of Cas9 used, the PAM sequence may be very common within the genome (i.e. spCas9’s 5’-NGG-3’), or not as common (i.e. saCas9’s 5’-NNGRRT-3’). In almost all cases a 5’-NGG-3’ PAM sequence will be present within the gene of interest.

TALENs are sensitive to CpG methylation, so design may require avoiding these sites. As well, TALENs must be used in pairs, since each nuclease domain only cuts one strand of DNA. This doubles the amount of design work required.

b. Experimental Workflow

Design of siRNAs is the easiest method, followed by design of sgRNAs for CRISPR and TALENs. In order to design siRNAs, one only needs the sequence of the corresponding mRNA transcript. Design for CRISPR and TALEN requires knowledge of the genomic DNA sequence.

i. Experimental Set Up

Because it uses host machinery to achieve silencing, RNAi has the simplest experimental set up and siRNA treatment can cause significant gene repression in only 24 hours (2). Only one transgene needs to be delivered into the cell. It can be prepared as a ~20 bp double-stranded siRNA, or a ~80 bp shRNA cloned into a vector.

In comparison, CRISPR and TALEN rely on exogenous nucleases that must be delivered into the cell. This limits their effectiveness for use with viral expression systems such as AAV (Adeno-Associated Virus), which have limited packaging capacity. Smaller alternatives to spCas9 (ex. saCas9 and Cpf1) have been developed to enable the use of AAV with CRISPR, but no such alternatives exist for TALEN.

The cloning of TALEN vectors is more difficult than cloning sgRNA or shRNA vectors because they are larger and contain repetitive sequences. As well, TALENs must be used in pairs, doubling the amount of cloning work that must be done.

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ii. Experimental Protocol

The time and labour involved in the gene silencing experiment will vary greatly depending on the delivery method chosen. It takes more time and effort for viral delivery than for plasmid or siRNA transfection since viral vectors must first be packaged. A selection marker can be used to enrich for cells that have taken up the plasmid, which will add additional time to the protocol, but may have better results.

iii. Experimental Validation

No matter the method used, gene silencing must be verified before conclusions can be made. The process for verifying gene silencing varies depending on the technique used.

When using RNAi, it’s best to use two validation methods: one measuring mRNA levels (such as qRT-PCR), and another measuring protein levels (such as Western blot). A decrease in mRNA levels seen without a corresponding decrease in protein levels indicates that protein turnover may be slow. A decrease in protein levels without a corresponding decrease in mRNA levels indicates that the siRNA may be exerting its effects via translational inhibition instead of mRNA degradation. The time required for these experiments is typically only a day or two, but an appropriate antibody for Western blot may not be available.

CRISPR and TALEN gene silencing can be verified with methods that target the DNA. Initial screening is usually performed using the Mismatch Cleavage Detection Assay (a.k.a. Surveyor or T7E1), which can within a day determine whether cells have undergone any editing and approximately how much. If partial gene knockdown is sufficient, these partially edited pools can be used directly for experiments.

If gene knockout is desired, monoclones must be isolated and screened for the presence of a biallelic frameshift mutation. Isolation usually is done by a process of serial dilution. Initial screening of monoclones can be done with the Mismatch Cleavage Detection Assay, but edited monoclones must be sequence verified to be sure that the edits cause a frameshift mutation. This is usually done with Sanger sequencing, adding days to the protocol. Check out our CRISPR Screening and Validation Knowledge Base for more information.

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c. Efficiency

The efficiency of any system varies depending on many factors including cell line, delivery mechanism, transfection/transduction efficiency, target site chosen, length of the siRNA/sgRNA/DNA-binding region, use of selection, etc. For this reason, it’s difficult to directly compare the efficiency of CRISPR, RNAi, and TALEN.

A large-scale screen of CRISPR and RNAi found that sgRNAs and shRNAs show similar on-target efficiencies (3). shRNAs tend to function less efficiently than siRNAs, perhaps because use of shRNA often results in fewer active molecules per cell (4). In comparison, TALEN usually shows lower efficiency than CRISPR and RNAi (5).

Efficiency is generally less important for gene knockout than for gene knockdown. A low efficiency when using CRISPR or TALEN means that more cells must be screened to find a knockout clone, but once that monoclonal cell line is found the gene will be completely silenced. In comparison, poor efficiency in a RNAi experiment will mean less gene repression, leading to less pronounced phenotypes.

d. Off-Target Effects

TALEN has the lowest number of off-target editing events because it requires two independent binding events to opposing DNA strands in order for editing to occur. The chance of another locus possessing both target sites is very low, so off-target activity by TALENs is usually minimal (11–13).

CRISPR can adopt a similar approach if off-target effects are a concern. One of Cas9’s nuclease domains can be mutated to make a Cas9 nickase, which will only cleave one strand of DNA. Like TALEN, two Cas9 nickases and two opposing target sequence sgRNAs are required for DNA cleavage. Use of Cas9 nickase has been shown to reduce off-targeting by 50-1500-fold (14, 15). Off-target effects caused by wildtype Cas9 are usually less than those caused by RNAi (3).

RNAi causes significant off-target effects. siRNA can induce silencing of non-target mRNAs with only limited sequence complementarity (16). One siRNA can potentially repress hundreds of transcripts (17) which can confound results. In addition, when a cell is flooded with exogenous short RNAs, they can displace endogenous miRNAs from the RISC. This impairs the normal functions of cellular miRNAs, leading to unintended effects (18). Even more concerning, multiple studies have shown that siRNAs can non-specifically trigger an interferon response in cells (19, 20). An interferon response results in a signalling cascade that activates many enzymes, including tyrosine kinases, signal transducers, and transcription factors. This not only makes it difficult to separate target-specific effects from off-target effects, but also raises concerns about using siRNA for therapeutic applications in vivo (21).

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d. Flexibility

CRISPR is the most versatile system for genetic manipulation, followed by TALEN and then RNAi. RNAi can only be used for gene silencing, while variations of CRISPR and TALEN can be used for gene knockdown, knockout, activation, repression, or base editing.

CRISPR can be easily adapted for gene knock-in, which can be used to add a reporter to an endogenous gene of interest. This can be achieved by providing a repair template that includes the reporter in addition to the other CRISPR Cas9 components. With the repair template present, the cell will undergo homology directed repair (HDR) and incorporate the new reporter sequence at the cut site.

CRISPR can also be adapted to perform other functions such as gene activation, repression, and imaging by using a mutant version of Cas9 that lacks cleavage capabilities (dCas9). dCas9 has the ability to specifically bind to a target region, but cannot cut DNA. By fusing dCas9 to effector proteins, the CRISPR Cas9 system can expand its role to gene activation (dCas9-SAM), gene repression (dCas9-KRAB), genome imaging (dCas9-GFP), and more. Similar effects have been achieved by using a TALE fused to an activator or repressor domain (22, 23).

The enzymes involved in miRNA regulation are present in many eukaryotic species, so RNAi can be used in plants, animals, nematodes, and yeast (24–26). However, these enzymes aren’t present in prokaryotes or viruses, so RNAi cannot be used in these organisms.

CRISPR has been used in a wide variety of eukaryotic organisms, as well. Because it relies on eukaryotic DNA repair mechanisms to prevent cell death, CRISPR cannot be used as-is in bacterial systems. However, it can be useful as a negative control method used during lambda red recombineering (27). TALEN has not been adapted in a similar way.

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d. Applications

Which gene silencing method is best to use will depend on the purpose of the experiment. If the purpose of the experiment is to discover the function of a gene by silencing it, gene knockout (CRISPR or TALEN) is preferred over gene knockdown (RNAi). Knocking out the gene will usually cause a more dramatic phenotype than if it was only knocked down, which translates into definitive results (28). Furthermore, CRISPR or TALEN can be used to introduce genetic mutations that more accurately resemble those that cause genetic diseases (29).

There are times, however, when a dramatic phenotype is not desired. For instance, if a gene is essential, a complete knockout will cause cell death. In these cases, it may be desirable to perform a RNAi experiment instead. RNAi may also be preferred if the intent is to mimic the effect of a drug, since inhibitive drugs will rarely completely inhibit a protein’s function. The reversible nature of gene knockdown can also be exploited to conditionally regulate a gene, simply by using a conditional promoter to express the shRNA (30).

There are some specialized applications which may require the use of gene editing over RNAi, or vice versa. For example, transcriptional repression via CRISPRi or TALE-KRAB can be used to study lncRNAs (long non-coding RNAs) where other techniques are not effective. lncRNAs aren’t translated so typical CRISPR and TALEN knockout via NHEJ aren’t effective, and lncRNAs reside in the nucleus so RNAi, which is mainly active in the cytoplasm, can’t be used.

In the case where the protein of interest degrades slowly in the cell, RNAi will not result in low protein levels. And even when protein levels are greatly reduced, if that gene product is not rate limiting, incomplete knockdown may not cause a loss-of-function phenotype (31).

High-throughput screening is an increasingly popular tool for functional genomics, as it allows one to collect large amounts of data at a genome-wide scale. Large-scale screening is best done using CRISPR or RNAi (32). Libraries of TALENs are challenging to make due to their large size, repetitive elements, and more difficult design requirements (33). In comparison, shRNA and sgRNA vectors can be designed and cloned more easily on a large scale, and several genome-wide or pathway specific libraries are commercially available for use. Or, RNAi and CRISPR can be used in concert – it has been found that a combinatorial approach can improve performance (34).

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Which gene silencing method is best to use will depend on many factors including the purpose of the experiment, the time and funds you have available for it, and how much of a concern off-target effects are.

Table 1 — Summary of Gene Silencing Methods.

Adapted from Boettcher and McManus (31).

If off-target effects are a particular concern, such as for therapeutics, TALEN or CRISPR using a Cas9 nickase should be considered since these have the greatest specificity. RNAi should be avoided since it has the most off-target effects.

Cost will vary for RNAi depending on whether siRNA oligos (expensive) or shRNA vectors (inexpensive) are used. CRISPR sgRNA vectors are a comparable price to shRNA vectors. TALENs tend to cost more due to their more complicated design and cloning requirements.

For ease of design and experimentation, RNAi is hard to beat. siRNAs and shRNAs are easy to design and don’t require the expression of any exogenous nucleases. CRISPR sgRNAs are also relatively easy to design and clone, but for complete gene knockout one must perform extra screening steps. TALENS are the most difficult to clone, and its low cleavage efficiency means there may be more screening required than for CRISPR.

In summary, the best gene silencing method to use will depend on the subject of research and practical concerns such as time and labour available. CRISPR is undoubtedly the most versatile technique, with variations that can achieve gene knock-in, activation, repression, and more. TALEN is able to cause gene knockout like CRISPR, but is less appealing due to its lower efficiency, difficult cloning, and large size. RNAi is limited to gene knockdown only, but is still a useful tool under circumstances where complete knockout is not desired.

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