Should I use CRISPR or TALEN for genome editing?
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.
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.
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.
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.
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.
In terms of 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. However, TALEN recognition modules, such the one built into the VectorBuilder system, have greatly reduced the work required to generate TALEN vectors. 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.