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CRISPR/Cas9 gene editing in hematology
specific Cas9 double-stranded DNA cleavage,10 illustrating the wide-ranging application of CRISPR as a genome-edit- ing technology.11 Indeed, the CRISPR/Cas9 system was first successfully used in human cells in 2013.12-14 The essential components of this technology include a gRNA that binds specifically to a 20-base pair sequence of inter- est, and the Cas9 enzyme – an endonuclease that intro- duces a DSB. Additionally, a conserved dinucleotide-con- taining protospacer adjacent motif sequence upstream of the gRNA-binding site is required by the endonuclease to recognize and cleave the sequence. In the case of Cas9 iso- lated from Streptococcus pyogenes, the most widely used nuclease, the protospacer adjacent motif sequence is NGG (Figure 2A). If these conditions are fulfilled, CRISPR/Cas9 can be directed to cleave any genomic sequence. Subsequently, the DSB (in eukaryotic cells) triggers endogenous cellular DNA-repair pathways that can be exploited either to generate gene knock-outs based on the introduction of insertions or deletions (indels) at the DSB by non-homologous end joining (NHEJ)15, or for genome editing, by introducing an engineered template DNA via homology-directed repair (HDR)16 (Figure 2A). In contrast to the protein-DNA interactions of other nuclease editing systems, CRISPR relies on Watson-Crick pairing between RNA and DNA. Thus, researchers keen to perform gene editing require only a basic knowledge of molecular biol- ogy to design a targeting system against a locus of choice.
Here, we will focus mainly on work done with the Cas9 nuclease, although it is worth mentioning that the CRISPR/Cas system can include many other enzyme vari- ants with numerous functions that are suitable for applica- tions beyond gene editing17 (Table 2).
In comparison with engineered nucleases, CRISPR/Cas9 is an easy-to-use genome-editing tool, and several differ- ent CRISPR/Cas9-component delivery methods are avail- able for in vitro, ex vivo and in vivo applications18 (Table 3). Generally, Cas9 and gRNA can be introduced into cells in several formats, such as plasmid DNA, lentiviral vectors, mRNA, or more recently by using pre-assembled ribonu- cleoprotein complexes (Table 4). Indeed, ribonucleopro- tein complexes are perhaps the best choice for clinical applications given their high efficiency and short window of action, which reduces the duration of nuclease expo- sure and, consequently, the possibility of undesired off- target effects. In the hematopoietic setting, CRISPR/Cas9 gene editing has been applied both in research and in clin- ical translation studies (Figure 2B). In disease modeling, CRISPR/Cas9 technology coupled to next-generation genomics allows researchers to faithfully recapitulate the genetic mutations seen in patients with clonal hematopoiesis or leukemia.19 In the clinical setting, the main goal is to employ CRISPR/Cas9 to treat diseases of the blood and immune system. With this view, several biotechnology companies have pipelines to develop and
Table 1. Pros and cons of genome engineering tools in mammalian systems.
Gene editing toolsa
Pre-edition era
Conventional gene edition
Meganucleases
ZFN
TALEN
CRISPR/Cas9
Advantages
• Genetic analysis relied on spontaneous, induced random mutations by chemicals or transposons
• DSB not usedb
• Different gene modifications: knock out,
conditional alleles, reporter genes.
• Large recognition site for DNA • Highly specific
• DSB repaired by HDR or NHEJ
•Possibilityofengineeringnucleases • Highly efficient
• DSB repaired by HDR or NHEJ
• Biallelic changes are possible
• Works in different cell types and species.
•EasiertodesignthanZFN
• Highly efficient
• DSB repaired by HDR or NHEJ
• Biallelic changes are possible
• Works in different cell types and species
• Easy design and optimization
• Highest efficiency
• DSB repaired by HDR or NHEJ
• Biallellic changes obtained with efficiency • Works in different cell types and species
Disadvantages
• Extremely laborious
• No directed gene editing
• Highly unpredictable mutations
• Extremely laborious
• Highly inefficient
• Large homology fragments of DNA are
needed for homologous recombination
• Biallellic changes are difficult to obtain
• Difficult to use in hESC and other cell types • Selection markers are necessary
• Very low design flexibility
• Low specificity of the enzyme/off-target
possibility
Main dates
1950s
1980s
1988
•Off-targeteffectpossiblebutlessthanwithCRISPR 1996 • Harder to design than TALEN nucleases
•Off-targeteffectpossiblebutlessthanwithCRISPR 2009 • Still harder to design than CRISPR
• More off-target effects than TALEN and ZFN 2013 (though there are ways to reduce them
dramatically)c
• PAM sequence limits target selection (though,
many CRISPR systems available, and more to come)d
aGene editing of mammalian genomes.bDSB:double-strand break;hESC:human embryonic stem cells;HDR:homology-directed repair;NHEJ:non-homologous end joining;PAM: protospacer adjacent motif; TALEN: transcription activator-like effector nucleases; ZFN: zinc finger nucleases. cThe use of nickases and/or ribonucleoproteins, which reduce the time window in which the nucleases can induce lesions,drastically reduces the probability of off-targets.dMany CRISPR systems have been described from different prokaryotes that use different PAM sequences.This allows for more flexibility when designing a targeting strategy.Relatively few CRISPR systems have been described to date.
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