Genome editing is a vital tool for scientists to be able to understand diseases, improve crops, and unlock the secrets of how our cells work, as well as many other applications. Up until about 5 years ago, genetic engineering usually meant weeks of time and effort, was expensive and had limited versatility. Now, similar experiments can be conducted in less than a week very, very cheaply. So, what changed? The introduction of CRISPR/Cas9 technology revolutionised genome editing. CRISPR (clustered regularly interspaced short palindromic repeats) meant that genes could be knocked out (deleted), inserted, or mutated easily, with high accuracy, at almost any position in the genome. Based off of a bacterial immune defence system, the CRISPR/Cas9 complex consists of several components. The Cas9 protein forms a complex with guide RNA that matches the sequence of the DNA to be targeted. The complex scans the genome for the protospacer adjacent motif (PAM) sequence, which is a short series of DNA bases immediately after the targeted DNA sequence. When it finds a match, the guide RNA pairs with the target DNA sequence and the Cas9 protein cuts the DNA in half, forming a double stranded break. Double stranded breaks in DNA are bad news for the cell, so the cell attempts to repair the cut. However, this process is prone to errors, so often mutations are introduced which means the gene no longer functions. Alternatively, scientists can introduce new genes by adding DNA which is integrated at the site of the cut. In this way, scientists can add or delete genes to study how they work. CRISPR/Cas9 is very versatile and efficient, but it’s not perfect. While it can target almost any region of the genome, it still needs that PAM sequence. The most commonly used protein recognises NGG (N being any DNA base and G being guanine), which is relatively short but still has to be present in the target sequence. CRISPR/Cas9 is also limited by off-target effects. It may accidentally cut other regions of the genome that closely match the guide RNA sequence and introduce unwanted mutations.
Recently, a group of researchers at Harvard set out to improve the Cas9 protein. They used a technique that forced the protein to rapidly evolve and accept a wider range of PAM sequences. They tested this new xCas9 variant with mammalian cells for compatibility and found that it will be suitable for a broad range of applications, especially in human cells. Surprisingly, xCas9 has greater specificity for the target DNA sequence, with fewer off-target effects observed than with the original Cas9. This illustrates that there is not necessarily a trade-off between specificity and versatility, and may help to improve future generations of Cas9 proteins. This improvement means CRISPR/Cas9 genome editing is even more versatile and will be able to target regions of the genome more specifically and more precisely, expanding the scope of genome editing and enhancing the potential of using CRISPR technology to understand and treat human disease.
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Emi Schutz Archives
March 2018
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