Synthetic theory of evolution

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ELEMENTARY EVOLUTION FACTOR
These are some factors which can lead to evolution

of species.
The four forces of evolution include:-
Mutation
Genetic drift
Gene flow
Natural selection

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MUTATION
The changing of the structure of a gene, resulting in a

variant form that may be transmitted to subsequent generations, caused by the alteration of single base units in DNA, or the deletion, insertion, or rearrangement of larger sections of genes or chromosomes

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GENETIC DRIFT
It is the variation in the relative frequency of

different genotypes in a small population, owing to the chance disappearance of particular genes as individuals die or do not reproduce.

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GENE FLOW
In population genetics, gene flow (also known as gene migration or allele flow) is the transfer

of genetic variation from one population to another. If the rate of gene flow is high enough, then two populations are considered to have equivalent allele frequencies and therefore effectively be a single population

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NATURAL SLECTION
The process whereby organisms better adapted to their environment

tend to survive and produce more offspring. The theory of its action was first fully expounded by Charles Darwin and is now believed to be the main process that brings about evolution

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Non-target is defined as non-specific genetic modifications as a result of a designed

genome editing process, including point mutations, deletions, insertions, inversions, and translocations of unexpected genes

NON-TARGETING MUTATION PROCESS

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MECHANISM
The CRISPR-Cas9 system works as the adaptive immune system in bacteria

and archaea.When a virus infects the bacteria, this system incorporates segments of the viral DNA into the bacterial genome. Upon a second invasion, transcripts from these sequences direct a nuclease activity to its complementary sequence in the invading virus so as to destroy it.
In order to extrapolate this method into eukaryotes in order to develop a gene editing method, a Cas9 protein, a recognition sequence RNA, and a transactivating RNA are required. The fusion of both the recognition sequence specificity CRISPR RNA (crRNA) and transactivating RNA (tracrRNA) is commonly used in experiments and called a single guide RNA (sgRNA).[ It performs both functions: the first 20 nucleotides of the sgRNA are complementary to the DNA target sequence (cr function), while the nucleotides following are part of a protospacer adjacent motif (PAM; tracr function).[

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Off-targeting nuclease binding originates from a partial but sufficient match to

the target sequence. Off-target binding mechanisms can be grouped into two main forms: base mismatch tolerance, and bulge mismatch.

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BASE MISMATCH TOLERANCE
While the Cas9 specificity is believed to be controlled

by the 20nt sgRNA and PAM, off-target mutations are still prevalent and could occur with as many as 3-5 base pair mismatches (out of 20) between the sgRNA and the target DNA sequence.[25][30] Furthermore, sgRNA secondary structures could also affect cleavage of on-target and off-target sites. As mentioned above, sgRNA consists of a sequence (~20nt) which is complementary to the target sequences and this is followed by a PAM sequence which activates the endonuclease activity. While it was shown that 10-12 nt adjacent to PAM (called the “seed sequence”) was enough for Cas9 specificity, Wu et al. showed that in a catalytically dead Cas9 only 1-5 base pairs of seed sequence is required for specificity.[31] This was later proven by other studies as well. The Cas9 protein binding is further affected by a number of mechanisms:
The seed sequence determines the frequency of a seed plus PAM in the genome and controls the effective concentration of Cas9 sgRNA complex.

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Uracil-rich seeds are likely to have low sgRNA levels and increase

specificity since multiple uracil in the sequence can introduce termination of the sgRNA transcription.[31][32]
Mismatches in the 5’ end of the crRNA are more tolerated as the important site would be adjacent to the PAM matrix. Single and double mismatches are also tolerated based on how to place it.
In a recent study, Ren et al. observed a link between mutagenesis efficiency and GC content of sgRNA. At least 4-6bp adjacent to the PAM are required for a good edit.[33]
While picking a gRNA, guanine is preferred over cytosine as the first base of the seed adjacent to PAM, cytosine as the first in the 5’ and adenine in the middle of the sequence. This design is based on stability linked to formation of G quadruplexes.[31][32][34]
A ChIP was performed by Kim et al. showcasing that addition of a purified Cas9 along with the sgRNA caused low off target effects which means that there are more factors causing these effects.[35]

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VALUE OF MUTATION IN EVOLUTION
Classical hypotheses of evolution have been derived

by a comparison of the morphological and other phenotypic properties of many different organisms. The more information was accumulated and carefully evaluated, the fewer hypotheses remained likely. More recently, evolution became explainable in molecular terms, owing to the rapidly accumulating knowledge of the possible alterations of DNA, the information transfer from DNA to RNA and proteins, and the chemical and functional properties of proteins. This knowledge allows one to state general rules about the evolution of macromolecules, rules that hold for both simple and complex organisms. Some hypotheses of evolution can thus be eliminated already by deduction from molecular principles rather than by induction from many phenotypic observations. A complete theory of evolution probably can be obtained only by a combination of both inductive and deductive reasoning

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This system provides an interesting case history in gene evolution. The

lactose-utilizing members of the group, such as Escherichia coli, have β-D-galactosidase and galactoside permease, plus galactoside transacetylase, determined by adjacent genes, z+ and y+, members of the lac operon. The absence of the permease and the resulting inability to use lactose effectively has played some remarkable evolutionary tricks on the rest of the lactose-utilizing gene set.

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The loss of permease has played the key role in the

evolution of the lac region by rendering the galactosidase inoperative in lactose utilization and has permitted the accumulation of a variety of mutations within the z gene.

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Proteins and genes might have required the simultaneous evolution of the

cell. Studies of the organismic evolution of proteins began with considerations of applying sequential and terminal residue methods to tracing evolution of primary Sructure of proteins.

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Some of the concepts that emerged from these studies were a

Darwinian explanation of micro-heterogeneity of protein preparations, a demonstration that the evolution of protein has proceeded to yield a minute fraction of the theoretical possibilities, an explanation of the similarities between protein molecules as related to slow stepwise substitution of residues, the use of such techniques in chemical taxonomy, and analyses of genealogy of homologous and heterologous proteins.

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EFFECTS OF MUTATION
Since all cells in our body contain DNA, there

are lots of places for mutations to occur; however, some mutations cannot be passed on to offspring and do not matter for evolution. Somatic mutations occur in non-reproductive cells and won't be passed onto offspring. For example, the golden color on half of this Red Delicious apple was caused by a somatic mutation. Its seeds will not carry the mutation.The only mutations that matter to large-scale evolution are those that can be passed on to offspring. These occur in reproductive cells like eggs and sperm and are called germ line mutations.

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