Canonical CRISPR-Cas9 genome editing technique has profoundly impacted the fields of plant biology, biotechnology, and crop improvement. Since non-homologous end joining (NHEJ) is usually considered to generate random indels, its high efficiency mutation is generally not pertinent to precise editing. Homology-directed repair (HDR) can mediate precise editing with supplied donor DNA, but it suffers from extreme low efficiency in higher plants. Therefore, precision editing in plants will be facilitated by the ability to predict NHEJ repair outcome and to improve HDR efficiency. Here, we report that NHEJ-mediated single nucleotide insertion at different rice genes is predictable based on DNA sequences at the target loci. Three mutation prediction tools (inDelphi, FORECasT, and SPROUT) have been validated in the rice plant system. We also evaluated the chimeric guide RNA (cgRNA) and Cas9-Retron precISe Parallel Editing via homologY (CRISPEY) strategies to facilitate donor template supply for improving HDR efficiency in Nicotiana benthamiana and rice. However, neither cgRNA nor CRISPEY improved plant HDR editing efficiency in this study. Interestingly, our data indicate that tethering of 200–250 nucleotides long sequence to either 5′ or 3′ ends of guide RNA did not significantly affect Cas9 cleavage activity.
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Diverse Systems for Efficient Sequence Insertion and Replacement in Precise Plant Genome Editing
CRISPR-mediated genome editing has been widely applied in plants to make uncomplicated genomic modifications including gene knockout and base changes. However, the introduction of many genetic variants related to valuable agronomic traits requires complex and precise DNA changes. Different CRISPR systems have been developed to achieve efficient sequence insertion and replacement but with limited success. A recent study has significantly improved NHEJ- and HDR-mediated sequence insertion and replacement using chemically modified donor templates. Together with other newly developed precise editing systems, such as prime editing and CRISPR-associated transposases, these technologies will provide new avenues to further the plant genome editing field.
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- NSF-PAR ID:
- 10270676
- Date Published:
- Journal Name:
- BioDesign Research
- Volume:
- 2020
- ISSN:
- 2693-1257
- Page Range / eLocation ID:
- 1 to 4
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract CRISPR‐Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA‐guided nuclease to initiate double‐strand breaks. Until recently, classical RAD51‐mediated homologous recombination has been a powerful tool for gene targeting in the moss
Physcomitrella patens . However, CRISPR‐Cas9‐mediated genome editing inP. patens was shown to be more efficient than traditional homologous recombination (Plant Biotechnology Journal, 15, 2017, 122). CRISPR‐Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR‐Cas9 genome editing inP. patens , here we describe the generation and use of a flexible and modular CRISPR‐Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology‐directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5′ and 3′ ends of gene coding regions. With regard to homology‐directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a “stop codon cassette” with downstream near‐effortless genotyping. -
CRISPR/Cas technology is increasingly being used as a common methodology in many cancer biology studies due to the ease and convenience of the technique. Precise editing of genomic DNA has been achieved upon repair of CRISPR-induced DNA double-strand breaks (DSBs) by homologous recombination (HR). HR repairs DNA DSBs with high fidelity and therefore, deficiencies in HR result in genome instability. These deficiencies have been demonstrated in many cancers. RAD51-dependent HR is a very important pathway for repairing DSBs. Previous studies have shown that genome editing using CRISPR technology relies on the repair of site-specific DNA DSBs induced by the RNA-guided Cas9 endonuclease. Furthermore, previous studies have shown that the efficiency of CRISPR-mediated HR can be improved by the stimulation of HR promoting factors, such as the RAD51 recombinase. Despite the ease and efficient use the CRISPR/Cas technology for genome editing, one limitation is the potential occurrence of associated off-target effects. If CRISPR technology is planned to be used to target cancer cells with defective HR capabilities, will off-target mutations be likely to occur? In order to answer this question, a system was developed in Saccharomyces cerevisiae using green fluorescent protein (GFP) as a reporter to identify off-target CRISPR-induced DSBs. This study set out to test the number of off-target DSBs that could be introduced by CRISPR-induced genome editing in a RAD51-deficient HR model. We were curious whether loss of RAD51-dependent HR would increase the abundance of off-target CRISPR-induced DSBs in mutant yeast strains as compared to those with a functioning HR-dependent DNA repair pathway. Preliminary findings using this system will be presented.more » « less
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Chia, Nicholas (Ed.)ABSTRACT A diversity of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide adaptive immunity to bacteria and archaea through recording “memories” of past viral infections. Recently, many novel CRISPR-associated proteins have been discovered via computational studies, but those studies relied on biased and incomplete databases of assembled genomes. We avoided these biases and applied a network theory approach to search for novel CRISPR-associated genes by leveraging subtle ecological cooccurrence patterns identified from environmental metagenomes. We validated our method using existing annotations and discovered 32 novel CRISPR-associated gene families. These genes span a range of putative functions, with many potentially regulating the response to infection. IMPORTANCE Every branch on the tree of life, including microbial life, faces the threat of viral pathogens. Over the course of billions of years of coevolution, prokaryotes have evolved a great diversity of strategies to defend against viral infections. One of these is the CRISPR adaptive immune system, which allows microbes to “remember” past infections in order to better fight them in the future. There has been much interest among molecular biologists in CRISPR immunity because this system can be repurposed as a tool for precise genome editing. Recently, a number of comparative genomics approaches have been used to detect novel CRISPR-associated genes in databases of genomes with great success, potentially leading to the development of new genome-editing tools. Here, we developed novel methods to search for these distinct classes of genes directly in environmental samples (“metagenomes”), thus capturing a more complete picture of the natural diversity of CRISPR-associated genes.more » « less
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Abstract Until recently, precise genome editing has been limited to a few organisms. The ability of Cas9 to generate double stranded DNA breaks at specific genomic sites has greatly expanded molecular toolkits in many organisms and cell types. Before CRISPR‐Cas9 mediated genome editing,
P. patens was unique among plants in its ability to integrate DNA via homologous recombination. However, selection for homologous recombination events was required to obtain edited plants, limiting the types of editing that were possible. Now with CRISPR‐Cas9, molecular manipulations inP. patens have greatly expanded. This protocol describes a method to generate a variety of different genome edits. The protocol describes a streamlined method to generate the Cas9/sgRNA expression constructs, design homology templates, transform, and quickly genotype plants. © 2023 Wiley Periodicals LLC.Basic Protocol 1 : Constructing the Cas9/sgRNA transient expression vectorAlternate Protocol 1 : Shortcut to generating single and pooled Cas9/sgRNA expression vectorsBasic Protocol 2 : Designing the oligonucleotide‐based homology‐directed repair (HDR) templateAlternate Protocol 2 : Designing the plasmid‐based HDR templateBasic Protocol 3 : Inducing genome editing by transforming CRISPR vector intoP. patens protoplastsBasic Protocol 4 : Identifying edited plants.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.34133/2020/8659064
