TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery

Abstract

Precise studies of plant, animal and human genomes enable remarkable opportunities of obtained data application in biotechnology and medicine. However, knowing nucleotide sequences isn't enough for understanding of particular genomic elements functional relationship and their role in phenotype formation and disease pathogenesis. In post-genomic era methods allowing genomic DNA sequences manipulation, visualization and regulation of gene expression are rapidly evolving. Though, there are few methods, that meet high standards of efficiency, safety and accessibility for a wide range of researchers. In 2011 and 2013 novel methods of genome editing appeared - this are TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems. Although TALEN and CRISPR/Cas9 appeared recently, these systems have proved to be effective and reliable tools for genome engineering. Here we generally review application of these systems for genome editing in conventional model objects of current biology, functional genome screening, cell-based human hereditary disease modeling, epigenome studies and visualization of cellular processes. Additionally, we review general strategies for designing TALEN and CRISPR/Cas9 and analyzing their activity. We also discuss some obstacles researcher can face using these genome editing tools.

Keywords: CRISPR/Cas9; TALEN; genome editing.

Fig. 1

A scheme for introducing a double-strand break using chimeric TALEN proteins. One monomer of the DNA-binding protein domain recognizes one nucleotide of a target DNA sequence. Two amino acid residues in the monomer are responsible for binding. The recognition code (single-letter notation is used to designate amino acid residues) is provided. Recognition sites are located on the opposite DNA strands at a distance sufficient for dimerization of the FokI catalytic domains. Dimerized FokI introduces a double-strand break into DNA

Fig. 2

A mechanism of CRISPR/Cas9 action in bacterial cells (see the text for details)

Fig. 3

A general scheme of the strategy for using the TALEN and CRISPR/Cas systems in genomic engineering

Fig. 4

A scheme of the modular hierarchical ligation strategy based on the Golden Gate cloning system to generate genetic constructs expressing chimeric TALEN proteins. A – at the first stage, a library of monomers is generated, which is a kind of a “construction kit” comprising a set of parts. These parts are amplified sequences of monomers with specific oligonucleotide primers. The primers are selected in such a way that hydrolysis by type IIS restriction endonucleases results in the formation of sticky ends, which define the monomer position in a final construct. B – a single Golden Gate reaction enables simultaneous ligation of multiple monomers, which results in intermediate k-mer constructs. C – at the last stage, the Golden Gate reaction is carried out, resulting in restriction and ligation of several intermediate k-mer constructs and the backbone plasmid containing the remaining TALEN elements

Fig. 5

Single chimeric sgRNA to introduce double-stranded breaks into the target loci. A complex of sgRNA and Cas9 is capable of introducing double-strand breaks into selected DNA sites. SgRNA is an artificial construct consisting of elements of the CRISPR/Cas9 system (crRNA and tracrRNA) combined into a single RNA molecule. A protospacer is a site that is recognized by the CRISPR/ Cas9 system. A spacer is a sequence in sgRNA that is responsible for complementary binding to the target site. RuvC and NHN are catalytic domains causing breaks at the target site of the DNA chain. PAM is a short motif (NGG in the case of CRISPR/Cas9) whose presence at the 3’-end of the protospacer is required for introducing a break

Fig. 6

A scheme of a genetic construct expressing CRISPR/Cas system elements. hCas9 is the Cas9 protein sequence optimized for expression in eukaryotic cells. sgRNA is a single chimeric RNA containing the parts of crRNA and tracrRNA necessary for the activity. NLS is the nuclear localization signal, which provides penetration of the construct into the nucleus. Poly (A) is the polyadenylation signal

Fig. 7

A scheme of various analyses to identify and determine the efficiency of double-strand break introduction caused by the TALEN and CRISPR/Cas systems. First, constructs encoding CRISPR/Cas9 or TALEN are delivered into cells. In cells, double-strand breaks occur in the target loci that are repaired by nonhomologous end joining (NHEJ). This results in the formation of insertions or deletions. Next, the target locus is amplified by PCR. PCR products are analyzed by the following methods. A – a target segment is cloned into a plasmid vector. Impairment or, instead, recovery of the reading frame of the lacZ gene occurs due to the insertions or deletions. Based on the count of blue and white colonies after the transformation of E. coli, the efficiency of the CRISPR/Cas9 or TALEN systems is determined; B – after cloning into a plasmid vector and E. coli transformation, Sanger sequencing is performed. Clones containing insertions/deletions are counted, the efficiency is determined; C – after denaturation and re-hybridization of the PCR product, DNA heteroduplexes are formed; e.g., one strand is “wild type,” and the other contains a deletion. After treatment with enzymes that cut DNA in unpaired segments, samples are loaded onto a gel and electrophoresis is carried out. The hydrolysis products mean that the sample contained heteroduplexes; hence, a break appeared in double-strand genomic DNA under the action of CRISPR/Cas9 or TALEN; D – a high resolution melting analysis enables heteroduplex detection. Blue is the control samples, red is the samples containing heteroduplexes; E – unpaired DNA regions reduce the heteroduplex mobility in a 15% polyacrylamide gel. After gel electrophoresis, bands corresponding to homo- and heteroduplexes can be observed