What kind of sparks will the “genetic scissors” encounter when jumping with a jumping gene?

Decoding gene editing

The breakup of Song Zhongji Song Hye Kyo of the performing arts industry makes people call: I only hope that I have never met. The encounters in the biological world are often irritating: seeing each other late - Zhang Feng, a "post-80s" scientist, became famous for inventing the most effective gene editing method and was called the father of CRISPR.

Barbara McClintock, many people are not familiar with, but her Cold Spring Harbor laboratory is considered to be the most active research destination. Barbara was the “zero after zero” (born in 1902) in the last century. In her time, people thought that the gene was “firmly anchored”, but she had to prove that the gene could “beat”, her Systematic research and adherence to science ultimately broke the conservative concept and earned the Nobel Prize in 1983.

The results of the research of two scientists across a hundred years have finally met. "Science" and "Nature" recently published two new CRISPR technologies, both of which improve the targeting accuracy of CRISPR technology by exploiting the nature of "jumping genes".

Can edit genes, they work like this when they are "single"

The CRISPR/Cas9 technology is the full name of the CRISPR technology. The former "CRISPR" is the "guide target", and the latter Cas9 is the real "scissor".

In the world of genes, "guide" must rely on base sequences. For CRISPR, it only recognizes one sequence that is clustered, regularly spaced, short palindrome, and repeated. The sequence of the Cas9 protein is cleaved by the guide RNA to identify the sequence that meets the requirements.

The cutting process has the dynamic characteristics of a small video. The Cas9 protein first forms a complex with the guide RNA, and the Cas9 protein undergoes a conformational change, like a piece of white paper rolled into a "cylindrical shape", which makes it easier for DNA to enter and exit. This soft complex will collide in the DNA. If the recognition is wrong, the "red light is on", the complex will leave and continue the next collision; if the recognition is correct, the "green light is on", the cut editing attempt is started.

Before the Cas9 protein begins to “moving scissors”, it needs to be re-checked. It unwinds the double-stranded DNA and uses the principle of RNA-DNA base pairing to further check the complementarity of the sequence and RNA. If there are more mismatches, the sequence identification process is terminated, ensuring that Cas9 is functioning at the correct target. If the identification is completed, the Cas9 is further activated and the scissors are activated.

The jumping gene is completely different. It is unconstrained, "hate" all dogmas, and the attributes of dogma such as "repetition" and "short essay" of CRISPR seem to be completely out of a "channel." It can “freely leave” from its own position, copy or break it alone, and “program” an annular ship for itself, “walk around”, encounter the right place, and insert another point.

Barbara found it because of the random changes in the shape of the corn it caused. She observed in Indian colored corn that there are often many spots on the kernels and leaves. The size of the stain, the appearance of the morning and evening, these changes, are controlled by the regulation of some unstable genes or "altered genes."

The jumping gene is a model of "liberalism", and it is difficult for people to grasp what can activate it. But the famous LINE1 is a major jumping gene. The "Cell" sub-publishes that scientists "catched its tail". Only in the presence of POLY-A (poly A) "tail" will LINE1 start. Jumping, without the "tail" jumping gene will be inactivated. In addition, transposases are key factors in the initiation of transposition. At present, some methods for guiding and controlling skipping genes by transposase have been mastered.

A pair of natural "CP", why are the two sides "complementary"?

The CRISPR/Cas9 gene editing technology faces problems such as low precision repair ratio, limited recognition sequence, and off-target phenomenon. According to the working mechanism of the gene "magic shear", it can be seen that the separate "guided positioning", melt-shearing, and complementary repair theory are all feasible.

The problem is that external interventions and influences are bound to be corrected by the system itself. "As long as the guide RNA is properly designed and positioned accurately, it is not difficult to accurately delete the corresponding DNA fragments." According to experts, but during the replacement process, the DNA repair system in the body will be activated. This repair system has high fault tolerance. It is possible to become a problem gene that can tolerate DNA damage.

However, the target gene carried by CRISPR needs to be homologously recombined to bind to the target genome. The homology recombination has a low fault tolerance rate and follows the principle of pairing. Therefore, it faces the “competition” of the repair system, resulting in low integration efficiency and even complete suppression. Imaginary off-target effects.

Sequence insertion into CRISPR-dependent homologous recombination, the "shifting" function of the transposon is more potent. The Tn transposon in bacteria is a piece of DNA containing several resistance genes and a gene encoding a transposase. Repeat or invert at both ends, corresponding to the site of action of the transposase. When bound to the target DNA, the transposome recognizes and attacks the target sequence to insert the transposon into the target sequence. The entire transposition process completes the process of the gene being spliced ​​from the original DNA and then affixed to another receptor DNA, realizing the "jumping" of the gene.

The new research work is to change the key Cas9 "scissors" and replace them with a new complex enzyme. According to reports, the researchers have established a CRISPR-related transposase, half of which is the CRISPR effector protein Cas12k, which is in line with Cas9, but only binds to DNA without cleavage, and is only responsible for localizing the target genome. The work of cleavage replacement by transposase By "taking over", the gene fragment can be inserted directly into the target site, completely eliminating the step of cutting the target genome.

It can be seen that the encounter over a hundred years is reflected in the improvement of key enzymes, through the integration and transformation of enzymes from different sources, so that the new Cas system is produced. According to the test results, the system can achieve a insertion success rate of up to 80% in the E. coli (prokaryotic) genome, which is much higher than the success rate of the corresponding editing of the CRISPR/Cas9 system.

In fact, this is not the first attempt to combine two methods of editing genes. According to reports, BMC Biology published a study in 2014. Scientists have discovered a new class of transposons in bacteria and archaea that contain not only the coding gene for Cas endonuclease. It also relies on the integration of this enzyme into new genomic regions, and scientists refer to this new transposon element as "Casposon." It can be seen that this pair of "CP" may have existed in nature.

Editing different species, improvement has been on the road

The gene editing CRISPR/Cas9 system was developed from the invasion of prokaryotes against foreign genetic components. The Tn7 transposon is also derived from the prokaryotic E. coli. The former grasped the direction and the latter carried out the actual operation.

In eukaryotic genes, how they perform, still need to be repeatedly tried, modified and verified.

The "mosaic phenomenon" is a problem that has been difficult to solve when the gene editing tool is applied to the editing of eukaryotic germ cells. "We used the microinjection method to edit the CRISPR gene in the fertilized egg, and hope to obtain the corresponding gene editing animal." Ji Yanxiao, assistant professor of research at Zhongnan Hospital of Wuhan University, said that the individual animals obtained by these methods may have editing cells at the same time. A chimeric individual with unedited cells. That is to say, some genetically edited mice may have only successful editing of the leg cells, or other parts, which is very influential in the construction of animal models.

"We can only screen out the next generation of transgenic animals to identify individuals that can be stably inherited, which is very time consuming and labor intensive for large animals," said Ji Yanxiao.

Yuan Weijun of Guangdong Medical University and others analyzed the "Progress in the Problems and Improvement Measures of CRISPR/Cas9 Technology". The occurrence of "mosaic phenomenon" may be due to the application of CRISPR/Cas9 system to the compilation of fertilized egg genes in multicellular organisms. The fertilized egg splits into different blastomeres, and the Cas9 protein has different editing and repairing methods for different blastomeres, resulting in chimeric individuals with different editing types of cells; or may be fertilized successfully to the genome first The time for secondary replication is extremely limited, and the “time window” left for genetic editing is very short.

To this end, the research work on improving the accuracy and reducing the mismatch rate of gene editing technology still needs to be continuously promoted to obtain optimal combinations and solutions for different species.

Source: Technology Daily

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