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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 mossPhyscomitrella patens. However, CRISPR‐Cas9‐mediated genome editing inP. patenswas 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.
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dCas9-based gene editing for cleavage-free genomic knock-in of long sequences
Abstract Gene editing is a powerful tool for genome and cell engineering. Exemplified by CRISPR–Cas, gene editing could cause DNA damage and trigger DNA repair processes that are often error-prone. Such unwanted mutations and safety concerns can be exacerbated when altering long sequences. Here we couple microbial single-strand annealing proteins (SSAPs) with catalytically inactive dCas9 for gene editing. This cleavage-free gene editor, dCas9–SSAP, promotes the knock-in of long sequences in mammalian cells. The dCas9–SSAP editor has low on-target errors and minimal off-target effects, showing higher accuracy than canonical Cas9 methods. It is effective for inserting kilobase-scale sequences, with an efficiency of up to approximately 20% and robust performance across donor designs and cell types, including human stem cells. We show that dCas9–SSAP is less sensitive to inhibition of DNA repair enzymes than Cas9 references. We further performed truncation and aptamer engineering to minimize its size to fit into a single adeno-associated-virus vector for future application. Together, this tool opens opportunities towards safer long-sequence genome engineering.
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- Award ID(s):
- 1953686
- PAR ID:
- 10338388
- Date Published:
- Journal Name:
- Nature Cell Biology
- Volume:
- 24
- Issue:
- 2
- ISSN:
- 1465-7392
- Page Range / eLocation ID:
- 268 to 278
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.more » « less
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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.more » « less
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The DNA inside human cells provides instructions for all of the processes that happen inside the body. Errors in the DNA may lead to cancer, sickle cell disease, cystic fibrosis, Huntington’s disease, or other genetic disorders. Medical researchers are exploring whether it is possible to replace or repair the faulty DNA (an approach known as gene therapy) to reduce the symptoms, or even cure individuals, of these conditions. Over the last ten years, a new technology known as CRISPR-Cas9 gene editing has proved to be a reliable and efficient way to make small and precise changes to DNA in living cells. First, an enzyme called Cas9 searches for a segment of target DNA segment that matches a template molecule the enzyme carries. Cas9 then cuts the target DNA, which is repaired to match a new customized DNA sequence: this changes the genetic information of the cell. The Cas9 protein is made of a succession of building blocks called amino acids that create long chains which then fold to form the final three-dimensional shape of the enzyme. A region of Cas9 known as the HNH domain is responsible for cutting the target DNA. However, it remains unclear exactly which amino acids within this domain work together to sever the DNA. Here, Zuo et al. combined computational and experimental approaches to reveal the three-dimensional structure of the Cas9 enzyme when the HNH domain is poised to cut the target DNA. The findings were used to generate a computational model of Cas9 and this model predicted that the HNH domain relies on a group of three amino acids known collectively as D839-H840-N863 to cleave DNA strands. This knowledge is useful to understand exactly how Cas9 modifies genetic information. Ultimately, this may help to improve CRISPR-Cas9 technology so it could be safely used in geneediting therapies.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1038/s41556-021-00836-1
