The latest genomic engineering tool used in science, CRISPR/Cas9 has been used in many studies. Research proves that it is efficient at crop improvement and helps prevent the spread of diseases. However, research exists that allow gene editing to be even more efficient with the use of multiple gRNAs (guide RNA). This review focuses on 3 experiments that generated multiple gRNAs on plants, mice, monkeys and trees to edit several genes at once. These results can be used to further our understanding of how to efficiently edit genes. It can also apply to humans by contributing to research about treatment for diseases.
Problems Presented
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat) is a tool used by researchers to modify specific DNA sequences. It is most commonly used for crop enhancement and molecular treatment. However, targeting limits exist due to the amount of gRNAs. gRNAs are responsible for directing Cas9 (CRISPR associated) enzyme to the DNA site of interest. Traditional methods of genomic engineering use microinjections and plasmid constructs. However, these methods are limited in terms of vector capacity and the type of system it can be used on. Thus, research done by Kabin developed a new method to produce several gRNAs at once. His team wanted to find out if making multiple gRNAs would allow Crispr/Cas9 to be more efficient at directing Cas9 to multiple target sites.
In previous studies, CRISPR/Cas9 has been used by injecting Cas9 and sgRNAs (single guide RNA) into pronuclear stage zygotes. Although this approach allows for specific DNA strands to be broken, it results in low knockout efficiency and shows mosaicism (editing in only some parts of cells). This caused researchers to find out if zygotic injection of several sgRNAs and Cas9 protein was sufficient to produce gene-modified species. Zuo and his team wanted to find out the efficiency of multiple sgRNAs in both monkey embryos and mice. Thus, researchers conducted serial crossbreeding to produce animals with complete gene deletion.
Although CRISPR/Cas9 has been used widely among many plant and animal, genome editing in woody plants is not as common. Thus, Fan tested gene editing and mutation targeting in the white chinese poplar, Populus tomentosa Carr. He wanted to find out if the CRISPR/Cas9 system is effective in trees as it is in other organisms. However, he also wanted to test the efficiency of DNA editing and mutagenesis targeting with the use of multiple sgRNAs. Thus, he used 4 sgRNAs to produce knockout mutations in woody plants.
- Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system (Kabin)
- One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs (Zuo)
- Efficient CRISPR/Cas9-mediated Targeted Mutagenesis in Populus in the First Generation (Fan)
Experimental Approaches and Results
Kabin began his study by using the tRNA-processing system to generate many gRNAs from a single transcript. His team chose this system since it is part of every living organism. Thus, they assumed that RNA production would be successful. After recognizing that RNase P and Z is able to cleave pre-tRNAs at specific sites to cut 5’ and 3’ sequences, they assumed that if gRNAs are included they will be cleaved in the same manner.
Researchers developed a polycistronic tRNA-gRNA gene (PTG). The gene structure had tandem repeats of tRNA-gRNA. They transfected rice protoplasts with PTG. Results showed that PTG overall increased the targeting ability of Cas9. The tRNA system efficiently recognized tRNA and removed gRNAs from the PTG transcript. These gRNAs then directed Cas9 to multiple target sites. Although it was found that PTG successfully generated multiple gRNAs, the PTG’s with fewer gRNA’s were more efficient at deleting DNA fragments at the four MAPK(mitogen-activated protein kinase) loci. Researchers also used the PDS (rice phytoene desaturase) gene and transgenic rice plants to assess the targeting ability at specific DNA sites in plants. Each approach confirmed the high efficiency of the PTG method.
In Zuo’s experiment, his team used green fluorescent protein(GFP)-targeting and the Cas9 protein to estimate the level of mosaicism in the zygote from wild type mice and homozygous Actin-EGFP males. Researchers designed 4 sgRNAs that targeted the exon of the GFP gene. At the blastocyst stage it was found that the blastocysts that used single-sgRNAs only some had a GFP signal and showed mosaicism. The blastocysts without gene editing were also GFP positive. When using multiple sgRNAS (2,3 or 4) he found that there were no GFP signals which showed that GFP was knocked out completely. The multiple sgRNAs also did not cause any harm to embryo development.
In order to find out if GFP knockout was indeed due to multiple sgRNAs, researchers injected multiple sgRNAs at a lower concentration. They found that there were no GFP signals again. When injected with 1 sgRNA at this lower concentration, GFP mosaicism was shown. This shows that GFP knockout was indeed due to the targeting of multiple sgRNAs rather than an increase in concentration of sgRNAs.
Researchers further used this approach to target the pigmentation gene Tyrosinase (Tyr) in the zygote stage of gene-edited mice. It was found that when using 3 or 4 sgRNAs, the mice were 100% albino. It also did not cause any harm to embryo development. This indicated that Tyr knockout was efficient.
In order to test if several genes can be knocked out in a single step by CRISPR researchers targeted the redundant genes mice have in DNA oxidation , Tet1, Tet2 and Tet3. They assayed 5-hydroxymethylcytosine (5hmC) level to determine the presence of Tet genes. When using 3 sgRNAs mosaicism showed and there was a high level of 5hmC. When researchers used 6 sgRNAs similar levels of 5hmC was found. Lastly, when using 9 sgRNAs no 5hmC was found which indicated that all Tet genes were knocked out efficiently due to multiple sgRNAs.
CRISPR/Cas9 was found to be successful not only in mice but also in monkey embryos of the monkey Macaca fascicularis. Researchers used 3 sgRNAs on monkey embryos to knock out the mutation Arntl and a portion of the circadian clock Prrt2. After injections of the sgRNAs, it was found that Arntl had 100% knockout efficiency while Prrt2 had 91%.
Lastly, Fan used 4 sgRNAs to target the DNA site which is used for chlorophyll biosynthesis PtoPDS (phytoene desaturase gene 8). Albino plants indicated mutation of PtoPDS thus, Fan wanted to find out if CRISPR/Cas9 was effective at knocking out the PDS gene. The sgRNAs complementary sites had a tandem repeat of guanosine nucleotides. The sgRNAs were inserted into cassettes which were later combined with Cas9 into a vector. It was found that after being transformed with the construct pYLCRIPSR/Cas9+sgRNA, 89% of the leaves developed at-least 1 shoot that was albino. This indicated that PtoPDS was efficiently lost in the poplars. The rate of albino mice occurred in more than 50% of the T0 transgenic poplar which is higher than what past studies on Arabidopsis have found. The poplar had at least 2 PDS genes which indicated that using multiple sgRNAs was able to efficiently knock-out multiple genomes at once.
To verify that the resulting plants were albino due to mutations of the PtoPDS gene, researchers selected clones of 8 random transgenic T0 plants. Over 100 clones were sequenced and showed that all of the plants with albino phenotype had a mutated PDS gene at the target sites. It was found that mutations occurred during repair mechanisms after Cas9 cleavage. Some alleles also had a sequence inversion between the targeting sites. This rearrangement ultimately caused PDS to be terminated in the poplars. It was also found that the highest amount of mutation occurred in target sites 2 and 3 with 89.3% efficiency.
Similarities/Differences
All 3 experiments focused on increasing the efficiency of CRISPR/Cas9. Although they tested their approach on different plants and animals, each researcher wanted to generate multiple gRNAs at once. Each researcher wanted to target several DNA sites and edit multiple genes at once. However, Kabin focused primarily on the PTG method to edit multiple genes in crops. While Zuo used zygotic injection to knock-out GFP in mice. He also tested knock-out efficiency in monkeys. unlike the other researchers, Zuo measured the levels of mosaicism during his experiment. Lastly, Fan developed multiple sgRNAs and tested on woody plants to knock-out the PtoPDS gene.
Conclusions and Suggestions
Kabin found that the endogenous tRNA processing system and PTG contribute to producing multiple gRNAs successfully. It was concluded that using multiple gRNAs increases mutagenesis targeting ability and allows for multiple genes to be knocked out at once. On the other hand, Zuo found that CRISPR/Cas9 was indeed a useful approach to edit genes in monkey and mice models. His experiment offered insight on a how to generate gene-edited mice and monkeys without mosaicism. Fan found that his method to use multiple sgRNAs was successful at proving that not only can it be used on plants and crops but it can also be used trees. Their method was also beneficial because did not cause any frameshift mutations of the target gene. Future researchers can use these results to efficiently correct genetic defects which can play a part toward treatment of various diseases.
2019-4-28-1556480858