Han Chunyu: What a better gene editing system than Cas9/sgRNA looks like.

Biotechnology Channel News: Genomic editing technology CRISPR/Cas9 has been listed by Science as one of the top ten scientific and technological advances of 2013.
CRISPR is short for regular interval clustered repeat sequences, cas is short for CRISPR-related proteins.
CRISPR/Cas was originally found in bacteria, a defense system used by bacteria to identify and destroy antiphages and other pathogen invasions.
In the CRISPR/Cas9 system, enzyme Cas9 is cut at a DNA target site determined as such: an RNA molecule called CRISPR RNA (crRNA) uses one of its sequences to pass through another RNA molecule called tracrRNA Base pairing binds together to form a chited RNA (tracrRNA/crRNA) and then, by using another part of the crRNA sequence to pair the base with the target DNA bit, this chited RNA can guide Cas9 to the target and cut.
In practical applications, tracrRNA and crRNA can be used as two guided RNAs (gRNAs) or fused together to form single-wizard RNA (single guide RNA, sgRNA) and used to guide enzyme Cas9 to bind to the target DNA sequence and cut, where Cas9, together with sgRNA, is called The Cas9-sgRNA system.
further research also confirms that CRISPR/Cas9's genome editing capabilities are only possible in the presence of short film segment DNA sequences called protospacer adjacent to the base sequence (protospacer adjacent motif, PAM).
cas9 can only be accurately cut if PAM is present near the DNA target site.
, the presence of PAM is also necessary to activate the enzyme Cas9.
Though scientists have optimized the CRISPR/Cas9 system in a variety of ways to improve its efficiency and specificity, there are still some shortcomings, such as the CRISPR/Cas9 system will still be cut at off-target sites (where there is a misallocation between gRNA and target DNA sequences), which can lead to many harmful consequences, such as cancer production;
in a new study, researchers from Hebei University of Science and Technology and Zhejiang University School of Medicine in China found that nucleic acid endoenzymes from the Argonaute protein family also use oligonucleotides as a guide to degrade invading genomes.
Specifically, they found that an Argonaute protein (NgAgo) from Natronobacterium Gregoryi, a nucleic acid endoenzyme, can be used to edit genomes in human cells, guided by guided DNA (guide DNA, gDNA).
study was published online May 2, 2016 in the journal Nature Biotechnology under the title "DNA-guided genome editing using the Natronobacterium Gregoryi Argonaute."
author of the paper is Associate Professor Chunyu Han, School of Biological Sciences and Engineering, Hebei University of Science and Technology.
Argonaute protein from Gerald's saline-based bacteria uses single-stranded gDNA to cut target DNA despite being from the Argonaute protein (TtAgo) from Thermus thermophilus or from Pyrococcus fu Riosus' Argonaute protein (PfAgo) is able to cut DNA target sequences in-body with phosphorylated single-stranded DNA at the 5' end, but they all require very high reaction temperatures, which prevents them from being used in mammalian cells.
to solve this problem, the researchers used the PSI-BLAST search tool to search the U.S. National Biotechnology Information Center's (NCBI) non-redundant protein sequence database using the PSI-BLAST search tool, targeting the TtAgo and PfAgo amino acid sequences, from which they identified a potential candidate protein: argonaute protein (or NgAgo) from the Strain of Alzheimer's SP2.
the researchers went on to design three 5'-end phosphate-rich, 24-nucleotide-long single-stranded gDNA, two single-stranded gDNAs that complement each other (remembered as FW and RV) and can be combined to the target of the pACYCDuet-eGFP mass;
addition, they designed a pair of 5'-end phosphate-occurring single-stranded gRNAs that can also bind to the same target.
the results confirm that NgAgo cannot cut the mass in the absence of gDNA or in NC gDNA.
when FW or RV gDNA is present, NgAgo is able to make intones of this super-helixed mass at 37 degrees C.
NgAgo was able to linearize this mass when both FW and RV gDNA were present.
however, neither NgAgo can cut this mass in the presence of single-stranded gDNA where phosphorylation does not occur at the 5' end or single-stranded gRNA at the 5' end.
, NgAgo is able to cut target double-stranded DNA at 37 degrees C when combined with single-stranded gDNA that is phosphorylated at the 5' end.
NgAgo binds to single-stranded gDNA and causes double-stranded fractures in target DNA To assess whether NgAgo can be used as a genome editing tool, the researchers first expressed NgAgo in 293T cells and studied whether it binds to endogenic nucleic acids in human cells. They did not detect nucleic acids combined with NgAgo purified from 293T cells, suggesting that single-stranded DNA with phosphorylation at the 5' end of human cells is very rare and that even if endogenous single-stranded DNA exists, NgAgo will not be directed to off-target levels.
When the researchers transduced 293T cells using the coding of NgAgo's mass particles and these synthetic single-stranded gDNA or gRNA (5'-end occurrence or no phosphorylation) of these synthetic 24 nucleotides, they found that NgAgo could only bind to single-stranded gDNA with phosphatification at 5' end, not single-stranded gDNA without phosphorylation at the 5' end.
When the mitochondria encoded NgAgo transdemed to 293T cells for 24 hours before transporting 5' end phosphorylated single-stranded gDNA to those cells, the researchers found a significant decrease in the number of NgAgo-binding single-stranded gDNA, suggesting that when NgAgo is expressed, it only loads single-stranded (or binding) gDNA in a relatively short period of time.
In line with this, when NgAgo, which is purified from 293T cells, does not load the single-stranded g-DNA at 37 degrees C, even if it is incubated in-body for up to 8 hours with the single-stranded gDNA, the single-stranded gDNA is not loaded onto NgAgo.
, single-stranded gDNA can only be loaded onto NgAgo at a relatively high temperature (55 degrees C) in in-body.
Only NgAgo, which has been purified from 293T cells that have been transfideed with single-stranded gDNA, can cut target DNA in-body, rather than NgAgo, which is incubated with single-stranded gDNA (FW gDNA, or RV gDNA) at 37 degrees C.
further experiments confirmed that NgAgo, which had been purified from 293T cells that had been transfed with NC gDNA, could not cut the target DNA even after incubation with FW gDNA for eight hours at 37 degrees C.
these results show that NgAgo is very faithful to its initial single-stranded gDNA and is not able to exchange single-stranded gDNA at 37 degrees C.
However, NgAgo is able to exchange single-stranded gDNA, i.e. reload single-stranded gDNA, at 55 degrees C, but NgAgo cannot cut target DNA as efficiently as it does when loading single-stranded gDNA at 37 degrees C, because a high temperature of 55 degrees C reduces NgAgo's enzyme activity.
addition, NgAgo removes several nucleotides from the target site of the double-stranded DNA (not single-stranded DNA) when cutting the target DNA.
researchers compared the editing efficiency of the NgAgo-gDNA system and Cas9-sgRNA system in mammalian cells and found that the NgAgo-gDNA system made the target gene insefficiency as efficient as the Cas9-sgRNA system.
this method, they also confirmed that for NgAgo, the optimal length of single-stranded gDNA is about 24 nucleotides.
NgAgo's ability to edit endogenic targets on the human genome with a wide range of genomic targets and low misalfatch tolerance.
, they modified NgAgo to attach a nuclear positioning signal to its amino end to ensure that NgAgo was in the nuclei of the cell.
they designed five single-stranded gDNAs targeting the 11th exosces of the human DYRK1A gene, which were then tested and sequenced by T7 nucleic acid endoenzyme I (T7EI) to confirm that all five gDNAs could guide NgAgo to efficiently cut the target DNA sequence.
researchers also tested 47 single-stranded gDNA targeting eight different genes and found that the genome editing efficiency of these gDNSes (21.3 to 41.3%) was 21.3 to 41.3 percent 21.3 percent, and did not observe NgAgo's apparent preference for sequences of a particular nature. The
researchers also tested the NgAgo-gDNA system in a variety of mammalian cell lineages, including breast cancer cell line MCF-7, human bone marrow cell line K562, and HeLa cell line, and found that in all of these cell line, NgAgo was able to efficiently induce double-stranded fractures at DYRK1A gene targets.
To study the effects of nucleotide mis-pairing of NgAgo activity between single-stranded gDNA and target DNA sequences, the researchers first designed a single-stranded gDNA with 24 nucleotides long for a DYRK1A gene target, and then introduced single-stranded gDNA mis matching at each location of the single-stranded gDNA.
They found that NgAgo-mediated target DNA cutting was very sensitive to single nucleotide misalification at each location of gDNA: cutting efficiency decreased by 73 to 100 percent, with single nucleotide misalification at P8 to P11 leading to the largest decrease in cutting efficiency (85 to 100 percent).
they also found that misalmed in any of the three consecutive positions would completely destroy the cut.
To study whether NgAgo was still sensitive to single nucleotide misalactitation when the length of single-stranded gDNA changed, the researchers synthesized single-stranded gDNA with 21 nucleotides long, and found that single-nucleotide misalification at any location in the shortened gDNA still significantly disrupted NgAgo activity;
considering that Cas9-sgRNA systems can tolerate even five nucleotide misplaces, these experiments have tentatively confirmed that the NgAgo-gDNA system is highly fidele.
researchers compared the efficiency of the DYRK1A gene target sites on the mammalian genome with ngAgo-gDNA and Cas9-sgRNA systems and found that the cutting efficiency was 20 (31.97 percent for the former and 32.2 percent for the latter).
, they also looked at whether NgAgo-gDNA was superior to Cas9-sgRNA in targeted cutting G-C-rich DNA bits.
, they confirmed that cas9-sgRNA was much less efficient at cutting HBA2 and GTA4 genes rich in G-CDNA sites than NgAgo-gDNA.
, the NgAgo-gDNA system has the potential to be used for a wider range of genomic stations than Cas9-sgRNA.
, Cas9, for cutting, must require the target site to be close to the upper reaches of the PAM, while Argonaute proteins such as NgAgo do not have such target sequence limits.
NgAgo accurately inserted DNA fragments into the genome to verify that NgAgo was able to target and cut the mammalian genome, the researchers tested whether the NgAgo-gDNA system could be used to edit the genome.
of the provider sequences mediated by homology-directed repair (HDR) is a widely used strategy that can be used to produce specific genomic modifications.
To do this, the researchers designed a fragment of the dna of a provider consisting of a reporting gene region and a G418 resistance gene, in which the reporting gene region consists of two reading boxes: one that encodes mRFP and the other that is a sequence of undying encoding eGFP, separated by the TGA terminator.
that this reporting gene region has no initiaters and therefore is not normally expressed.
After transfecting the coding of NgAgo's protons, single-stranded gDNA, and fragments of the provider's DNA into 293T cells, the researchers detected cells that expressed mrRFP and confirmed that in those cells, the supply's DNA fragments were accurately inserted into the desired location.
G418 screening, they isolated a cell clone strain containing fragments of the provider's DNA.
, the researchers used the NgAgo-gDNA system to target the cutting of this positive cytoske.