Cas9 Nuclease Is a Precise Tool for Genome Editing

Introduction

Cas9 genome editing has rapidly become one of the most widely used tools for altering genomes in vivo. Cas9 genome editing is often referred to as CRISPR or CRISPR-Cas9, referencing the bacterial genome element from which it was developed, but in fact, only the Cas9 component is used in editing. Cas9 nucleases are encoded in the genomes of most bacteria and archaea, where they are usually adjacent to a CRISPR locus, clustered regularly interspaced short palindromic repeats.

As the mechanistic details of Cas9 function were discovered, two research groups, one led by Jennifer Doudna and Emmanuelle Charpentier and the other by Feng Zhang, sought to adapt Cas9 for genome editing. In this process, genomic DNA can be directly modified and the procedures are general enough to be used for any cell into which DNA can be introduced and expressed.

DNA recognition features of Cas9 nuclease

Like restriction enzymes, Cas9 is an endonuclease that cuts both strands of a target DNA. However, there is an important difference between the two types of nucleases, in terms of how they recognize their target sequence in double-stranded DNA. Restriction enzymes recognize four to eight base pairs through contacts between the DNA molecule and amino acid side chains in the enzyme. Cas9, however, is a ribonucleo protein consisting of a polypeptide and a guide RNA (gRNA). Recognition of target DNA for cleavage occurs by hybridization of about 20 bases between the gRNA and its complementary DNA sequence in the genome.

In microbes, the CRISPR locus is the source of the gRNA, and the Cas9 nuclease protects the cell from viral attack. Sequences in the CRISPR locus derive primarily from mobile genetic elements (bacteriophage and plasmids), so the Cas9 nuclease in a microbial cell specifically targets invading DNA for destruction.

The extreme specificity conferred by the gRNA is the key to genome editing because each 20-base target sequence is almost certainly unique, even in a large eukaryotic genome. In contrast, a restriction enzyme that recognizes a few nucleotides will cut the genome, on average, every few thousand bases. Cas9 enzymes can be engineered to carry gRNAs with specified nucleotide sequences, thereby programming the recognition sequence for the nuclease. The gRNA directs Cas9 to hybridize with a single, desired site in a genome, making it the most precise mechanism available for targeting and cutting DNA.

In eukaryotes, all of which lack a CRISPR/Cas system, the editing process begins by introducing the two components of the mature Cas9 endonuclease, the apoenzyme and the gRNA, to the host cells. These molecules may be added directly, or they may be added as cloned DNA regulated by an inducible promoter. In the latter case, upon induction, the Cas9-gRNA complex assembles and performs its DNA cutting function.

 

 

How Cas9 recognizes and hydrolyzes a specific DNA sequence. A portion of the gRNA protrudes from the enzyme, available for hybridization. Upon locating its complement, the gRNA induces a conformational shift in the nuclease (protein) portion of Cas9, which then hydrolyzes phosphodiester bonds in both DNA strands. In the simplest case, a point mutation is created as the cell scrambles to repair the damage.

Some microbes and all eukaryotes have a nonhomologous end joining (NHEJ) system that can rejoin the two chromosome pieces. But because it’s not a perfect repair, the fused DNA ends often have a deletion or insertion of a few base pairs. The consequence is usually a frameshift mutation in the gene that will result in an inactive protein. A limitation to this method is that the outcome differs in each cell.

A precise Cas9 genome edit changes the nucleotides to a predetermined sequence (e.g., a mutated gene could be replaced with the wild-type allele). In this case, a donor sequence must be constructed in vitro using seamless cloning techniques. The donor DNA includes chromosomal regions flanking the gRNA binding site to provide regions of homology for recombination with the chromosome. The donor sequence is introduced to the cell at the same time as the Cas9/gRNA molecules.

Homologous recombination between the broken chromosome and the donor sequence will repair the double-stranded break and at the same time incorporate the DNA carried on the donor molecule into the genome. Selection and screening procedures to identify mutants are not required with Cas9 gene editing, unlike traditional cloning. Cells into which the Cas9-gRNA complex has been introduced can survive and reproduce only if the original target sequence is modified. Unrepaired double-stranded chromosome breaks are lethal.

Although the molecular components of Cas9 editing originally derived from the bacterium Streptococcus pyogenes,
this technique has been used most frequently in eukaryotic systems.

There are several reasons for this:

• Bacteria (~45%) and most archaea (~85%) have homologues of the Cas9 editing system, complicating its use in these organisms.
• Other tools for genome editing already exist for many microbes.
• Most bacteria lack a NHEJ repair pathway for repairing the double stranded break generated by Cas9 nuclease and cannot survive this treatment, but homologous repair can be used efficiently.

Despite these challenges, Cas9 editing has been used successfully in some microorganisms that are difficult to manipulate genetically.

Applications for Cas9 nuclease

Researchers have developed other applications for Cas9 nuclease that take advantage of its ability to bind chromosomal DNA in a single genomic location. An altered version of Cas9, termed dead Cas9 (dCas9), retains the DNA localization function conferred by the gRNA, but no longer cuts DNA. Instead, a dCas9-gRNA molecule bound to DNA can serve as a platform for other enzymes. In the simplest example, dCas9-gRNA bound to a promoter acts as a repressor to inhibit expression of target genes.

Conversely, to activate transcription, dCas9-gRNA has been fused to the ω subunit of RNA polymerase so it can bind to the promoter of a target gene and recruit RNA polymerase. Genome editing has been demonstrated using dCas9-gRNA fused to additional DNA modifying enzymes.

Because the dCas9 variant is used, there is no nuclease activity to break the phosphate-sugar backbone, but the other enzymes directly modify the base, for example, from C to T. The outcome is a precise change to the DNA sequence. Experiments like these have demonstrated the versatility of the dCas9-gRNA module as a homing device to direct a range of effector molecules to a specific chromosomal location.

Cas9 gene editing is a relatively new technique that holds much promise, and scientists are actively exploring modifications and improvements. Projects as varied as removing allergens from peanuts to curing genetic diseases in humans have been proposed as examples of what might be achievable. Importantly, Cas9 technology opens the prospect of engineering the human genome in germ line cells (e.g., egg cells or sperm cells), thereby inviting discussions on many fronts to address ethical concerns.

Reference and Sources

• https://pharmaceuticalintelligence.com/tag/mirna/
• https://www.researchgate.net/publication/264433647_Structural_basis_of_PAMdependent_target_DNA_recognition_by_the_Cas9_endonuclease
• https://www.oatext.com/crispr-cas9-system-a-revolution-in-gene-editing.php
• https://www.frontiersin.org/articles/10.3389/fpls.2016.01740/full
• https://www.researchgate.net/publication/11577650_Recombination_at_doublestrand_breaks_and_DNA_ends_conserved_mechanisms_from_phage_to_humans
• https://www.jyi.org/2018-november/2018/11/1/the-biology-of-native-and-adapted-crispr-cas-systems
• https://www.sciencedirect.com/science/article/pii/S0167779917303049
• http://badgerapbiology.weebly.com/uploads/1/2/7/2/12727137/restriction_enzyme_lab.pdf
• https://www.researchgate.net/publication/348628647_CRISPRCas9_System_a_Revolutionary_Tool_in_the_Fight_Against_Antimicrobial_Resistance

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• Classification of viruses on the basis of genome
• Comparison Between the Domains Bacteria, Archaea, and Eukarya
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• Reverse Transcription Polymerase Chain Reaction (RT-PCR)