Abstract
SummaryThe CRISPR/Cas9 system and related RNA‐guided endonucleases can introduce double‐strand breaks (DSBs) at specific sites in the genome, allowing the generation of targeted mutations in one or more genes as well as more complex genomic rearrangements. Modifications of the canonical CRISPR/Cas9 system from Streptococcus pyogenes and the introduction of related systems from other bacteria have increased the diversity of genomic sites that can be targeted, providing greater control over the resolution of DSBs, the targeting efficiency (frequency of on‐target mutations), the targeting accuracy (likelihood of off‐target mutations) and the type of mutations that are induced. Although much is now known about the principles of CRISPR/Cas9 genome editing, the likelihood of different outcomes is species‐dependent and there have been few comparative studies looking at the basis of such diversity. Here we critically analyse the activity of CRISPR/Cas9 and related systems in different plant species and compare the outcomes in animals and microbes to draw broad conclusions about the design principles required for effective genome editing in different organisms. These principles will be important for the commercial development of crops, farm animals, animal disease models and novel microbial strains using CRISPR/Cas9 and other genome‐editing tools.
Highlights
Clustered regularly interspaced short palindromic repeats (CRISPRs) are repetitive sequences found in bacterial and archaeal genomes interrupted by spacers captured from previously encountered virus genomes and other invasive DNA
In one of the first examples of CRISPR/Cas9 genome editing, Li et al (2013) failed to induce homology-dependent repair (HDR) in Arabidopsis protoplasts and attributed this to the intrinsically low efficiency of HDR in these cells compared to N. benthamiana, but more recent data suggest that the low efficiency probably reflected the relatively small number of double-strand breaks (DSBs), given that a 1.1%–5.6% mutation frequency was achieved by nonhomologous end joining (NHEJ) with the same synthetic guide RNA (sgRNA)
The CRISPR/Cas9 system has been used for genome editing in a wide range of different organisms but the outcome in terms of resolution, efficiency, accuracy and mutation structure depends on various factors including target site choice, sgRNA design, the properties of the endonuclease, the type of DSB introduced, whether or not the DSB is unique, the quantity of endonuclease and sgRNA, and the intrinsic differences in DNA repair pathways in different species, tissues and cells
Summary
The CRISPR/Cas system and related RNA-guided endonucleases can introduce double-strand breaks (DSBs) at specific sites in the genome, allowing the generation of targeted mutations in one or more genes as well as more complex genomic rearrangements. Much is known about the principles of CRISPR/Cas genome editing, the likelihood of different outcomes is species-dependent and there have been few comparative studies looking at the basis of such diversity. We critically analyse the activity of CRISPR/Cas and related systems in different plant species and compare the outcomes in animals and microbes to draw broad conclusions about the design principles required for effective genome editing in different organisms. These principles will be important for the commercial development of crops, farm animals, animal disease models and novel microbial strains using CRISPR/Cas and other genome-editing tools
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