Genome Editing-the ability to make specific changes at targeted genomic sites-is of fundamental importance to researchers in biology and medicine. Nowadays, three programmable nucleases have been developed for genome editing, including zinc finger nuclease (ZFN), transcription activator-like e-ector nuclease (TALEN), and CRISPR (clustered regularly interspaced short palindromic repeats). ZFNs and TALENs comprise a powerful class of tools. These two chimeric nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a non-specific DNA cleavage domain, and enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone non-homologous end joining or homology-directed repair at specific genomic locations.
Compared with ZFNs and TALENs thchnologies, CRISPR-Cas system shows the highest specificity and efficiency and is the simplest one to implement and quickly become the most popular and powerful tool for genome engineering. CRISPR system not only provides a molecular tool for investigating biological questions in depth, but also enables the development of innovative and practical applications of biology. With the rapid development of ZFN, TALEN and CRISPR (CRISPR-Cas9 system, in particular) technologies, manipulating genome becomes relatively easy and efficient.
ZFN: Zinc finger proteins (ZNFs) were the first of the "genome editing" nucleases to hit the scene. ZFN consists of a zinc finger DNA-binding domain and DNA cleavage domain of FokI, the most thoroughly studied type IIS restriction endonuclease. Zinc fingers are the most common DNA binding domain found in eukaryotes. They typically are comprised of ~ 30 amino acid modules that interact with nucleotide triplets. Each ZNF typically recognizes 3-6 nucleotide triplets. Because the nucleases to which they are attached only function as dimers, pairs of ZNFs are required to target any specific locus: one that recognizes the sequence upstream and the other recognizes the sequence downstream of the site to be modified. Since this editing system needs to be synthesized commercially and is difficult to use, ZFN was gradually replaced by other systems.
Fig 1. The working mechanism of ZFN, TALEN and CRISPR
TALEN: TALEN is another engineered nuclease, which shows better specificity and efficiency than ZFN. Similar to ZNFs, TALENs use DNA binding motifs to direct the same non-specific nuclease to cleave the genome at a specific site, but instead of recognizing DNA triplets, each domain recognizes a single nucleotide. The interactions between the TALEN-derived DNA binding domains and their target nucleotides are less complex than those between ZNFs and trinucleotides, and designing TALENs is generally more straightforward than ZNFs. However, the major challenge for TALEN is to clone the large modules in series, joint these modules in designed order by ligase in an efficient way. And the other technique barrier is low screening efficiency for successful targeted cells. Both techniques have been engineered in many species, and are not limited to mouse embryonic stem cells.
CRISPR: The latest exciting development in gene editing technology is the CRISPR-Cas system. CRISPR systems are RNA-based bacterial defense mechanisms designed to recognize and eliminate foreign DNA from invading bacteriophage and plasmids. They consist of a Cas endonuclease that is directed to cleave a target sequence by gRNA. Both the Cas nuclease and gRNA are encoded by a "CRISPR locus" in the bacterial genome, and the system can be co-opted to cleave any target sequence by modifying the sequence of gRNA. Similar to the ZNF and TALEN systems, the CRISPR-Cas system can be used to introduce either random mutations at the site of DNA cleavage by non-homologous end joining or specific mutations or insertions by co-injecting an engineered DNA construct with homology to the DNA on either side of the cleavage site.
Genome editing starts with efficient DSB generation in the target DNA. Once the DSB is introduced, DNA repair can be achieved via two different mechanisms: the high ﬁdelity homology-directed repair (HDR) or, in the absence of a homologous repair template, via the error-prone non-homologous end joining (NHEJ). A DSB is usually repaired by NHEJ in most situations. NHEJ often results in variable lengths of insertion and deletion mutations (indels), and thus can be used to knockout genes. when single- or double-stranded DNA templates with homologous sequences that correspond to sequences ﬂanking the break site are introduced within the cell, the lesion may be repaired using the HDR machinery.
The CRISPR-Cas system offers several advantages over the ZNF and TALEN mutagenesis: a. Target design simplicity. Because the target specificity relies on ribonucleotide complex formation and not protein/DNA recognition, gRNAs can be designed readily and cheaply to target nearly any sequence in the genome specifically. b. Efficiency. Modifications can be introduced by directly injecting RNAs encoding the Cas protein and gRNA into developing mouse embryos. c. Multiplexed mutations. Mutations can be introduced in multiple genes at the same time by injecting them with multiple gRNAs.
|Source||Bacteria, Eukaryotes||Eukaryotes||Bacteria (Streptococcus sp.)|
|DNA binding determinant||Zinc ﬁnger protein||Transcription-activator-like effector||crRNA/sgRNA|
|Binding speciﬁcity||3 Nucleotides||1 Nucleotide||1:1 Nucleotide pairing|
|Mutation rate (%)||10||20||20|
|Target site length (bp)||18–36||24–40||22|
|Endonuclease||Fok I||Fok I||Cas9|
|Double-stranded break pattern||Staggered cut (4–5 nt, 5′ overhang)||Staggered cut (Heterogeneous overhangs)||Sp Cas9 creates blunt ends; Cpf1 creates staggered cut (5′ overhang)|
|Ease of design||Difﬁcult||Moderate||Easy|
|Best suited for||Gene knockout, Transcriptional regulation||Gene knockout, Transcriptional regulation||Gene knockout, Transcriptional regulation, Base editing|
|Applications||Human cells, pig, mice, tobacco, nematode and zebraﬁsh||Human cells, water ﬂea, cow and mice||Human cells, wheat, rice, maize and Drosophila|
All three technologies offer researchers with alternative methods to develop mutant mice and human disease models, faster than traditional gene targeting methods, but they also have some limitations and complications, such as, off-site effects: mutation introduced at non-specific loci with similar, but not identical, homology to the target sites are one of the most important complication of these technologies; mosaicism: mice with a mutant allele in only some of their cells can be produced; and multiple alleles: Healing of the nuclease cleavage site by non-homologous end joining can produce cohorts of mice with different mutations from the same targeting constructs, requiring genome sequencing to verify the nature and position of the specific mutation.
Despite these difficulties, ZNFs, TALENs and especially the CRISPR-Cas systems are powerful new tools for manipulating the genomes of not only mice but also somatic and embryonic stem cells from other species, including humans. It is likely the refinements of these systems will continue and that they will be adapted in new ways to create ever more sophisticated animal models for and genetic therapies for treating human diseases. Together, these technologies promise to expand our ability to explore and alter any genome and constitute a new paradigm to understand and treat diseases.
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