CRISPR Genome Editing Solutions

Either shRNA-mediated knockdown or nuclease-mediated knockout (e.g. CRISPR or TALEN) can be valuable experimental approach to study the loss-of-function effects of a gene of interest in cell culture. In order to decide which method is optimal for your specific application, there are a few things you should consider.

Mechanisms

Knockdown vectors

Knockdown vectors express short hairpin RNAs (shRNAs) that repress the function of target mRNAs within the cell by inducing their cleavage and repressing their translation. Therefore, shRNA knockdown vectors are not associated with any DNA level sequence change of the gene of interest.

Read more about our shRNA knockdown vectors 

Knockout vectors

CRISPR and TALEN both function by directing nucleases to cut specific target sites in the genome. These cuts are then inefficiently repaired by the cellular machinery, resulting in permanent mutations, such as small insertions or deletions, at the sites of repair. A subset of these mutations will result in loss of function of the gene of interest due to frame-shifts, premature stop codons, etc. If two closely positioned cut sites in the genome (i.e. within several kb) are targeted simultaneously, this can also result in the deletion of the intervening region.

Read more about our CRISPR vectors 

Effectiveness

shRNA-mediated knockdown will never completely repress the expression of the target gene. Even for the most effective shRNAs, some residual expression of the target gene will remain. In contrast, in a fraction of treated cells, CRISPR and TALEN can generate permanent mutations which may result in complete loss of gene function.

Consistency and uniformity

shRNA vectors generally provide high cell-to-cell uniformity within the pool of treated cells and very consistent results between experiments. In contrast, CRISPR and TALEN produce results that are highly non-uniform from cell to cell due to the stochastic nature of the mutations introduced. To fully knock out the gene of interest in a cell, all copies of the gene in the cell must be knocked out. Given that normal cells have two copies of any gene (except for X- or Y-linked genes) while cancer cells can have more than two copies, such full knockout cells may represent a very small fraction of all the treated cells. For this reason, nuclease-mediated knockout experiments require the screening of clones by sequencing to identify the subset in which all copies of the gene of interest have been knocked out.

Off-target effects

Off-target effects have been reported for both shRNA-mediated knockdown and nuclease-mediated knockout. The off-target phenotype(s) can be estimated by using multiple different shRNAs to target the same gene. If a gene knocked down by multiple different shRNAs results in consistent phenotype(s), then it argues against the phenotype(s) being caused by off-target effects. For CRISPR- or TALEN-mediated knockout, multiple clones containing loss-of-function mutations should be analyzed in order to account for any phenotype(s) that may be due to off-target mutations. Additionally, bioinformatically identified off-target sites could be sequenced in the clones to see if they have been mutated.

Design your homologous recombination donor vector online 

Both CRISPR and TALEN systems have been harnessed to edit genomes of cultured cells and model organisms. Both systems can be used to knock out genes, or to knock in point mutations or insertions, but these two systems are different in several ways and have their own pros and cons.

Mechanisms

CRISPR

The CRISPR system uses a site-specific guide RNA (gRNA) to direct the Cas9 nuclease to its target site in the genome to create DNA cleavage. The target sequence is typically ~20 bp long, and sites containing a few mismatches may still be recognized and cleaved.

Read more about our CRISPR vectors 

TALEN

The TALEN system employs a pair of chimeric proteins, each composed of a TAL effector DNA-binding domain (recognizing a specific sequence) fused to a FokI nuclease domain. The pair of proteins are designed to bind to a pair of target sites in the genome, each ~18 bp long and flanking a 14-20 bp spacer. Upon binding to DNA, the Fokl nuclease domains on the pair of proteins are able to dimerize, which in turn leads to DNA cleavage within the spacer region between the two target sites.

Efficiency

Both CRISPR- and TALEN-mediated genome editing show good efficiency, but the efficiency varies a lot depending on application, species and cell type. In general, CRISPR can be delivered into cells and induce DNA cleavage more efficiently than TALEN.

Off-target effects

A CRISPR gRNA targets ~20 bp sequence, whereas a TALEN pair binds to a total of ~36 bp target sequence. In addition, Cas9/gRNA complex has higher tolerance for sequence mismatches (up to 5 bp mismatches) than TALEN does. Therefore, TALEN-mediated cleavage has better specificity than CRISPR, and off-target cleavage in the genome by TALEN is unlikely. In contrast, off-target effects have been reported for CRISPR in cell lines, though analyses of CRISPR knockout mice suggest lower off-target frequency in vivo. Recent developments of CRISPR system have significantly enhanced CRISPR specificity. By using Cas9 nickase (Cas9 mutant that contains only one catalytic nuclease domain, e.g. Cas9_D10A and Cas9_H840A) with dual gRNAs, two single-strand DNA nicks are generated with close proximity of the target region, resulting in a double-nick DSB (double-strand break) within the target region that could be repaired. In this design, the off-target effects are minimized since the dual gRNAs expand the target sequence to ~40 bp long.

Target site requirements

TALEN can be generated to specifically target nearly any sequence in the genome. In contrast, target site selection for CRISPR is limited by the requirement for a PAM sequence (typically NGG) sequence located on the immediate 3’ end of the gRNA target sequence. This is no barrier to knocking out genes because cleavage anywhere in the gene is potentially effective but may present difficulties in generating site-specific mutations or insertions that require cleavage at a specific position of the gene. To precisely edit a specific genomic site using CRISPR, a homologous recombination donor vector or long oligo containing the desired edit sequence flanking by the immediate upstream and downstream homology arms of the target site can be delivered to the cells together with gRNA(s) and Cas9, in order to guide HDR (homology directed repair)-mediated DNA repair at the target site.

Design your homologous recombination donor vector online 

Technical challenges

In terms of technical simplicity, CRISPR out competes TALEN in several ways. First, for vector construction, CRISPR system only needs to construct a short gRNA because targeting of Cas9/gRNA complex relies on simple RNA/DNA hybridization, while TALEN system requires re-engineering of the TAL DNA-binding domain that is unique for each protein-DNA interaction. Therefore, gRNAs are cheaper and easier to design and construct than TALENs which always require two vectors per target site. Secondly, for some applications, such as injecting mouse embryos, Cas9 protein and gRNA can be more efficiently delivered via direct injection, but TALEN cannot. Thirdly, CRISPR is extremely versatile in genetic screening experiments since CRISPR screening library expressing many thousands different gRNAs can be easily constructed in a high-throughput manner.

For CRISPR-mediated genome editing, Cas9 nuclease is directed to the target site of site-specific guide RNA (gRNA) in the genome to create DNA cleavage. In most cases, to generate simple gene knockout, a single gRNA can be used together with Cas9 to generate a double-strand break (DSB), which is then inefficiently repaired by the non-homologous end joining (NHEJ), resulting in permanent mutations, such as small insertions or deletions, at the site of repair. A subset of these mutations will result in loss of function of the gene of interest due to frame-shifts, premature stop codons, etc.

Dual gRNAs can be used if Cas9_D10A nickase is being used to target the two opposite strands of a single target site. In this approach, the nickase enzyme will generate single strand cuts on both strands, one guided by each of the two gRNAs, resulting in DSBs at the target site. Generally, this method reduces off-target effects of CRISPR/Cas9 expression because targeting by both gRNAs is necessary for DSBs to be generated.

Dual gRNAs can also be used when Cas9_D10A nickase and an exogenous donor DNA template are being used to introduce specific base-changes (e.g. knockins) into a gene of interest. In this approach, the two opposite strands would be targeted by the two gRNAs at two sites flanking the desired mutation site, and homology-directed repair (HDR) pathways make use of the exogenous donor template to repair the excised sequence.

View options of our dual gRNA vectors