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CRISPR-based genome editing through the lens of DNA repair.
Genome editing technologies operate by inducing site-specific DNA perturbations that are resolved by cellular DNA repair pathways. Products of genome editors include DNA breaks generated by CRISPR-associated nucleases, base modifications induced by base editors, DNA flaps created by prime editors, and integration intermediates formed by site-specific recombinases and transposases associated with CRISPR systems. Here, we discuss the cellular processes that repair CRISPR-generated DNA lesions and describe strategies to obtain desirable genomic changes through modulation of DNA repair pathways. Advances in our understanding of the DNA repair circuitry, in conjunction with the rapid development of innovative genome editing technologies, promise to greatly enhance our ability to improve food production, combat environmental pollution, develop cell-based therapies, and cure genetic and infectious diseases.
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Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations.
The programmable sequence specificity of CRISPR has found uses in gene editing and diagnostics. This manuscript describes an additional application of CRISPR through a family of novel DNA enrichment technologies. CAMP (CRISPR Associated Multiplexed PCR) and cCAMP (chimeric CRISPR Associated Multiplexed PCR) utilize the sequence specificity of the Cas9/sgRNA complex to target loci for the ligation of a universal adapter that is used for subsequent amplification. cTRACE (chimeric Targeting Rare Alleles with CRISPR-based Enrichment) also applies this method to use Cas9/sgRNA to target loci for the addition of universal adapters, however it has an additional selection for specific mutations through the use of an allele-specific primer. These three methods can produce multiplex PCR that significantly reduces the optimization required for every target. The methods are also not specific to any downstream analytical platform. We additionally will present a mutation specific enrichment technology that is non-amplification based and leaves the DNA in its native state: TRACE (Targeting Rare Alleles with CRISPR-based Enrichment). TRACE utilizes the Cas9/sgRNA complex to sterically protect the ends of targeted sequences from exonuclease activity which digests both the normal variant as well as any off-target sequences.
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CRISPR-based approaches for studying inborn errors of immunity.
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Comment on the article by Zaami S, et al. "CRISPR-based techniques: Cas9, Cas13 and their applications in the era of COVID-19". Eur Rev Med Pharmacol Sci 2021; 25 (3): 1752-1761.
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CRISPR-Cas9 Based Genome Editing in Wheat.
The development and application of high precision genome editing tools such as programmable nucleases are set to revolutionize crop breeding and are already having a major impact on fundamental science. Clustered regularly interspaced short palindromic repeats (CRISPR), and its CRISPR-associated protein (Cas), is a programmable RNA-guided nuclease enabling targeted site-specific double stranded breaks in DNA which, when incorrectly repaired, result in gene knockout. The two most widely cultivated wheat types are the tetraploid durum wheat (Triticum turgidum ssp. durum L.) and the hexaploid bread wheat (Triticum aestivum L.). Both species have large genomes, as a consequence of ancient hybridization events between ancestral progenitors. The highly conserved gene sequence and structure of homoeologs among subgenomes in wheat often permits their simultaneous targeting using CRISPR-Cas9 with single or paired single guide RNA (sgRNA). Since its first successful deployment in wheat, CRISPR-Cas9 technology has been applied to a wide array of gene targets of agronomical and scientific importance. The following protocols describe an experimentally derived strategy for implementing CRISRP-Cas9 genome editing, including sgRNA design, Golden Gate construct assembly, and screening analysis for genome edits. © 2021 The Authors. Basic Protocol 1: Selection of sgRNA target sequence for CRISPR-Cas9 Basic Protocol 2: Construct assembly using Golden Gate (MoClo) assembly Basic Protocol 3: Screening for CRISPR-Cas9 genome edits Alternate Protocol: BigDye Terminator reactions for screening of CRISPR-Cas9 genome edits.
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CRISPR/Cas: a toolkit for plant disease diagnostics.
Genetic factors and infectious pathogens that cause plant diseases have a major impact on agricultural production. In recent years, the potential of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system in nucleic acid analysis and plant disease diagnostics has been demonstrated. We highlight progress of CRISPR/Cas technology that is significant for monitoring plant growth and preventing diseases.
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A CRISPR response to pandemics?: Exploring the ethics of genetically engineering the human immune system.
Ethical challenges should be addressed before gene editing is made available to improve the immune response against emerging viruses.
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Gene Editing for Duchenne Muscular Dystrophy Using the CRISPR/Cas9 Technology: The Importance of Fine-tuning the Approach.
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At Last: Gene Editing in Human Embryos to Understand Human Development.
Our understanding of early human development is typically based on inference from animal models, which may not fully recapitulate human embryonic features. As proof of concept, Fogarty et al. (2017) used CRISPR/Cas9 to genetically ablate the OCT4 gene in human preimplantation embryos and found key differences from its function in model systems.
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Advances in CRISPR/Cas-Based Strategies on Zoonosis.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) has emerged as the predominant technique for gene editing technique due to its high efficiency and low cost. In the area of zoonosis, CRISPR/Cas is also widely used in different research areas. This paper reviewed the principles of CRISPR/Cas technique and its applications in zoonosis. Moreover, we analyze the shortcomings and weaknesses that currently limit its use, highlight its direction for improvement, and foresee its application prospects in the prevention and treatment of zoonosis. For the purpose of preventing and controlling zoonosis, we need to develop diagnostic method with high sensitivity and specificity, highly protective vaccines, and also better understanding of the pathogenesis. Our review aimed to promote the application and improvement of CRISPR/Cas technique in the above-mentioned areas, and provide brief and comprehensive references for CRISPR/Cas-based research. Through reviewing the advances in CRISPR/Cas-based strategies on zoonosis, we believe that CRISPR/Cas technique will provide more powerful assistance for the prevention and control of zoonosis.
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