2018/02/25

Scientists start to realize that mutations don't drive evolution

Natural selection is not able to weed out harmful mutations - Scientists try to revert genetic alterations

https://www.genomeweb.com/gene-silencinggene-editing/broad-institute-labs-develop-new-crispr-systems-point-mutations-rna

Excerpt: "NEW YORK (GenomeWeb) – Broad Institute researchers David Liu and Feng Zhang have both developed new CRISPR-based systems, one for editing point mutations in the genome and the other for editing RNA, they revealed today in separate studies.

Liu's work, published in Nature, uses a guide RNA and catalytically impaired CRISPR-Cas9 to convert A-T base pairs to G-C base pairs in the genome, enabling the editing of single point mutations without the induction of double-stranded DNA breaks (DSBs).

The study builds on previous work done in Liu's lab in which researchers developed third-generation base editors called BE3s that consist of a catalytically impaired CRISPR-Cas9 mutant that cannot make DSBs, a single-strand-specific cytidine deaminase that converts C to uracil in the single-stranded DNA bubble created by Cas9, a uracil glycosylase inhibitor that impedes uracil excision and downstream processes that decrease base editing efficiency and product purity, and nickase activity to nick the non-edited DNA strand, directing cellular mismatch repair to replace the G-containing DNA strand.

In a press conference call about the study, Liu said this kind of editing enables the efficient and permanent conversion of C-G base pairs to T-A base pairs, and addresses point mutations found in roughly 15 percent of genetic diseases."

My comment: Actually more than 50% of disease-causing genetic mutations are missense/nonsense point mutations. (http://www.hgmd.cf.ac.uk/ac/index.php)

"However, the spontaneous deamination of cytosine and 5-methylcytosine in DNA is a major source of de novomutations, and about half of known pathogenic SNPs are C-G to T-A transitions, Liu and his coauthors at the Broad and Harvard University wrote in their paper. Therefore, converting A-T base pairs to G-C bases could help treat many more genetic diseases.

The deamination of adenine results in inosine, which is treated as guanine by DNA and RNA polymerases. However, there are no naturally occurring enzymes that deaminate adenine in DNA, so the researchers' first task was to directly engineer such an enzyme. They engineered E. coli TadA tRNA adenosine deaminase (TadA) through seven rounds of bacterial evolution to accept DNA as a substrate when fused to a catalytically impaired CRISPR-Cas9.

What resulted was an Adenine Base Editor (ABE). It transforms A to inosine, which is read as G. Similar to the BE3 base editor, the ABE also contains a nickase which causes the non-edited DNA strand to replace the T with a C. The CRISPR-Cas9 contained in the system binds to the guide RNA, and guides the ABE to the editing site — however, it has been impaired so that it can no longer induce DSBs.

REPAIRing RNA nucleotides


Meanwhile in Science, Zhang and his coauthors at the Massachusetts Institute of Technology and Harvard Medical School described their new CRISPR-based RNA editing system, called RNA Editing for Programmable A to I Replacement (REPAIR), which allows for the temporary repair of single RNA nucleotides in mammalian cells without permanently altering the genome.

While DNA editing holds promise for treating genetic disease, temporary editing of RNA has potential for treating diseases caused by temporary changes in cell state. In their study, the researchers profiled Type VI CRISPR systems to engineer a Cas13 ortholog capable of robust knockdown, and used catalytically-inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 to transcripts in mammalian cells.

To demonstrate the broad applicability of the REPAIR system for RNA editing in mammalian cells, the team designed guides against two disease-relevant mutations: 878G>A in X-linked nephrogenic diabetes insipidus and 1517G>A in Fanconi anemia. They transfected expression constructs for cDNA of genes carrying these mutations into HEK293FT cells and tested whether REPAIR could correct the mutations, and found that they were able to achieve 35 percent correction in the first and 23 percent correction in the second.
 

They then tested the ability of REPAIR to correct 34 different disease-relevant G-to-A mutations and found that they were able to achieve significant editing at 33 sites with up to 28 percent editing efficiency. "The mutations we chose are only a fraction of the pathogenic G-to-A mutations (5,739) in the ClinVar database, which also includes an additional 11,943 G-to-A variants," the authors wrote. "Because there are no sequence constraints, REPAIRv1 is capable of potentially editing all these disease-relevant mutations, especially given that we observed editing regardless of the target motif."

In the press conference for his Nature paper, Liu called Zhang's work "an impressive and exciting development," and said that he's hopeful that DNA base editing and RNA base editing can be used together as complementary tools to address a broad range of research and therapeutic applications."

My comment: The cell is able to edit sequences of RNA-molecules. This complex mechanism is called RNA editing. Scientists try use this designed mechanism for reverting pathogenic point mutations.

A few years ago SNP's were thought to be the main reason for different human traits. Today we know that methylated cytosines are prone to turn into thymines in deamination caused by oxidative stress and other factors contributed by diet or life habits. Today scientists are understanding that the DNA is not determining traits of organisms. They are also realizing that mutations result in genetic degradation and loss of biological information. Serious scientists also understand that the cell uses the DNA as a passive information library for building RNA-products, that mediate the necessary information for cellular differentiation, metabolism, immunity and transmission of environmental signals. That's why it's a good idea to edit RNA-molecules. Editing DNA might not be successful because the DNA is never used directly in cellular processes. A functional RNA is not a direct copy of a DNA strand. It needs modification and it is regulated by several epigenetic factors, such as chromatin shape, CpG methylation and histone markers.

There is no natural selection that could eliminate these pathogenic mutations. Genetic diseases are in an explosive growth and scientists are in a hurry to develop efficient methods for repairing human genome. The theory of evolution is the most serious heresy of our time. Don't get lost.