For years scientists have been touting the potential of CRISPR, a gene editing tool that allows you to target a specific mutation and either cut it out or replace it with the corrected form of the gene. But like all new tools it had its limitations. One important one was the difficult in delivering the corrected gene to mature cells in large numbers.
Scientists at the Gladstone Institutes and U.C. San Francisco say they think they have found a way around that. And the implications for using this technique to develop new therapies for deadly diseases are profound.
In the past scientists used inactivated viruses as a way to deliver corrected copies of the gene to patients. We have blogged about UCLA’s Dr. Don Kohn using this approach to treat children born with SCID, a deadly immune disorder. But that was both time consuming and expensive.
CRISPR, on the other hand, showed that it could be easier to use and less expensive. But getting it to produce enough cells for an effective therapy proved challenging.
The team at Gladstone and UCSF found a way around that by switching from using CRISPR to deliver a double-stranded DNA to correct the gene (which is toxic to cells in large quantities), and instead using CRISPR to deliver a single stranded DNA (you can read the full, very technical description of their approach in the study they published in the journal Nature Biotechnology).
Alex Marson, MD, PhD, director of the Gladstone-UCSF Institute of Genomic Immunology and the senior author of the study, said this more than doubled the efficiency of the process. “One of our goals for many years has been to put lengthy DNA instructions into a targeted site in the genome in a way that doesn’t depend on viral vectors. This is a huge step toward the next generation of safe and effective cell therapies.”
It has another advantage too, according to Gladstone’s Dr. Jonathan Esensten, an author of the study. “This technology has the potential to make new cell and gene therapies faster, better, and less expensive.”
The team has already used this method to generate more than one billion CAR-T cells – specialized immune system cells that can target cancers such as multiple myeloma – and says it could also prove effective in targeting some rare genetic immune diseases.
The California Institute for Regenerative Medicine (CIRM) helped support this research. Authors Brian Shy and David Nguyen were supported by the CIRM:UCSF Alpha Stem Cell Clinic Fellowship program.
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Prokaryotes have no intron but only have exons in their genome. Whereas eukaryotes have both introns and exons in their genome. As a result, in eukaryotes, when mRNA is transcribed from DNA, the introns have to be cut out of the newly synthesized mRNA strand. In eukaryotes, DNA is transcripted into RNA, which is edited in the nucleus. However, prokaryotes has no nucleus but nucleoles which leads transcription couple to translation without introns splicing that may stop genetic expression. Intron in eukaryotes have important roles 1) allowing alternative splicing to generate multiple proteins from a single gene. 2) some introns encode functional RNA molecules through further processing after they are spliced. 3) possibility of creat new genes by cutting and pasting exons from existing gene, or to diversify the protein output of a single gene by splicing the exons together in different ways. 4)splicing is necessary to create mRNA molecules that are capable of being translated into proteins.
Therefore, CRISPR-Cas9 is efficiency tool to edit DNA in prokaryotes. In humans, there are 26,564 annotated genes which consist of 233,785 exons and 207,344 introns in their genome . On average, there are 8.8 exons and 7.8 introns pergene. Any attempt to remove or alter the introns of gene might break the traditional pathway of transcription -splicing leading to the production of abnormal proteins which may harm the health and lives of people. Current investigation showed that improved protocol of CRISPR-Cas9 genome editing can be reach to about 80-90% of efficiency. The small number of incorrected editing cells cannot be taken lightly, they may be produced uncertainty or negative dominant effect, which may harmful to the health and safety of patients.