Category: CRISPR-Cas9

The Multiple Application of CRISPR-Cas9

CRISPR-Cas9 gene editing technology can target virtually any genomic location of choice by utilizing a short RNA guide, then cut the sequence with the Cas9 protein. Compared with traditional gene editing technologies, the CRISPR-Cas9 system is both faster and more efficient. The CRISPR-Cas9 system currently has a wide variety of applications in gene engineering and bio-medicine, such as gene editing in model organisms and cell cultures to further investigate the relationship between genetic variation and biological function. Other examples of CRISPR-Cas9 utilization include cultivation of new, robust seeds that are more resistant to extreme environments, or transgenically modified corn to enable the production of low-cost fuels.

  1. Genetic and epigenetic control of cells through genome engineering technologies is enabling a broad range of applications, from basic biology to biotechnology and medicine.
  2. Causal genetic mutations or epogenetic variants associated with altered biological function or disease phenotypes can now be rapidly and efficiently recapitulated in animal or cellular models.
  3. Manipulating biological circuits could also facilitate the generation of useful synthetic materials, such as algae-derived, silica-base diatoms for important agricultural crops to confer resistance to environmental deprivation or pathogenic infection, improving food security while avoiding the introduction of foreign DNA.
  4. Sustainable and cost-effective biofuels are attractive sources for renewable energy, which could be achieved by creating efficient metabolic pathways for ethanol production in algae or corn.
  5. Direct in vivo correction of genetic or epigenetic aberrant variation in somatic tissue could be a permanent genetic solution to genetically encoded disorders.
  6. Finally, engineering cells to optimize high yield generation of drug precursors in bacterial factories could significantly reduce the cost and accessibility of useful therapeutics. In addition to precise gene, the CRISPR-Cas9 system has many other potential applications, such as genome screening, endogenetic gene transcription, and genomic loci imaging.

The History of CRISPR-Cas9

The CRISPR (Clustered regularly interspaced short palindromic repeats) system was first identified in archaea as an adaptive defensive mechanism that confers resistance to foreign genetic elements. Later on, the CRISPR-Cas system was engineered into a versatile gene-editing tool enabling manipulation of protospacer adjacent motif (PAM) downstream DNA. Now, CRISPR-Cas9 has facilitated robust genome editing in virtually any organism including: human cells, rat, mice, zebra fish, bacteria, fruit flies, yeast, nematode and etc.

In 1987, Japanese scientists discovered a set of 29nt repeats interspaced by five intervening 32nt non-repetitive sequences in the Escherichia coli genome. The body of interspaced repeat sequences from different bacterial and archaeal strains is quickly expanding, and the nomenclature of microbial genomic loci consisting of an interspaced repeat array was unified as CRISPR (Clustered regulatory interspaced short palindromic repeats) in 2002. Over the next few years, CRISPR-Cas9 technology has been rapidly and widely adopted by the scientific community to target, edit, and modify the genomes of a vast array of cells and organisms while elucidating and refining the mechanism of CRISPR-Cas9 library genome editing. CRISPR-Cas9 has been harnessed for applications in screening for drug targets, human gene therapy, and pathogen gene disruption.

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The CRISPR-Cas9 System

CRISPR is a ubiquitous family of clustered repetitive DNA elements present in 90% of Archaea and 40% of sequenced Bacteria. The 300-500bp leader located upstream of CRISPR loci is a conserved, AT-rich sequence, and is considered a promoter of CRISPR array. CRISPR array consists of repetitive sequences (repeats) interspersed with several variable sequences (spacers). Repeats are typically 21–48 nucleotides in length with the potential to form hairpin structures. The variable spacer sequences in CRISPR array are derived from previous invading mobile genetic elements (MGEs), e.g. bacteriophages and plasmids. Prokaryotes with CRISPR-Cas immune systems capture short invader sequences within the CRISPR loci in their genomes, and small RNAs produced from the CRISPR loci (crRNAs) guide Cas proteins to recognize and degrade (or otherwise silence) the invading nucleic acids. Complete genome sequencing studies showed the presence of common sequences flanking the multiple CRISPR loci in multiple prokaryotic species. Comparison of the genes that flank the CRISPR loci in the genomes of different species showed a clear homology among those genes, which was later designated as CRISPR-associated genes, Cas. Cas genes encode proteins with a variety of nucleic acid-manipulating activities such as nucleases, helicases and polymerases, and are often located adjacent to the CRISPR region.

 

What are the advantages of CRISPR-Cas9?

The CRISPR-Cas9 system is perhaps the most remarkable recent breakthrough in genome editing technology. CRISPR is a ubiquitous family of clustered repetitive DNA elements present in 90% of Archaea and 40% of sequenced Bacteria. CRISPR arrays consist of interspersed identical REPEAT sequences (21-48bp) and several unique invader-targeting SPACER sequences (26-72bp). The CRISPR-Cas9 genome editing system consists of two components: a “guide” RNA (gRNA) and a non-specific CRISPR-associated endonuclease (Cas9). The Cas9 protein is an endonuclease that uses gRNA to form base pairs with DNA target sequences, enabling Cas9 to introduce a site-specific double-stranded break in the DNA. Through RNA-directed Cas9 nucleases, the CRISPR-Cas9 system can modify DNA with greater precision than existing technologies like TALEN and ZFN.

An advantage the CRISPR-Cas9 system offers over other mutagenic techniques like ZFN and TALEN is the relative simplicity of its plasmid design and construction. For each target site, the specificity of CRISPR-Cas9 relies on the formation of a ribonucleotide complex of sgRNA and the target DNA as opposed to protein/DNA recognition. CRISPR-Cas9 is easily programmable by changing the guide sequence (20 nucleotides in the native RNA) of the sgRNA to any DNA sequence of interest. Additionally, CRISPR is capable of modifying chromosomal targets with high fidelity whereas ZFN/TALEN are prone to CpG methylation. Last but not least, multiplexed genome editing with CRISPR-Cas9 library can be easily achieved with the monomeric Cas9 protein and any number of different sequence-specific gRNAs. The simplicity of CRISPR-Cas9 programming and its capacity for multiplexed target recognition have fueled the popularity of this cost-effective and easy-to-use technology.