NEW YORK (GenomeWeb) – New results from genetic screening firm Cellecta demonstrate that structural changes to single guide RNAs (sgRNAs) used in CRISPR/Cas9-based screens can increase gene knockout efficiency.
Cellecta announced the results, from a validation study of structural changes to CRISPR/Cas9 single guide RNAs (sgRNAs), last week. The study was funded by a Phase I small business innovation research (SBIR) grant from the National Institutes of Health, under which Cellecta received $224,905 over six months to develop a human genome-wide sgRNA library screen.
With the grant, scientists at the firm tested several changes to the part of the sgRNA that is not programmed to match the genomic target. They found that two changes, a single nucleotide switch just a few base pairs away from the targeting portion and a five nucleotide insertion in a structural portion that interacts with the Cas9 enzyme, were able to improve the efficiency of cellular dropout viability screens.
The firm was so confident the changes would work, it had already incorporated them into its sgRNA library products, Donato Tedesco, Cellecta's lead research scientist, told GenomeWeb. The new results are just the vindication of that decision. "We made a bet and we won the bet," he said.
Cellecta, which has been selling short hairpin RNA libraries for RNAi screens for years, began offering CRISPR/Cas9 screening about two years ago.
"CRISPR has exploded for us in the last 12 months," Tedesco said. While the firm offers screening services as well as library products, pooled CRISPR sgRNA libraries have overtaken shRNA libraries in popularity.
But genome-wide screens place a premium on the knockout efficiency of each component. "For high-throughput screening applications, it is very important that you have the minimum amount of no knockout, because that gives you a lot of noise in your system," Tedesco said, adding that knockout efficiency above 90 percent for every gene is an important threshold.
Increasing the efficiency of sgRNAs by 5 percent is equivalent to a two-fold improvement in the system, he said. "A genetic screen can work or not work depending on whether you are above or below the 90 percent knockout efficiency. The efficiency of gene editing is there at the border where a knockout can show up or not show up in a genetic screen. Any improvement that makes it go over that threshold could make the screen successful."
Prior to applying for the SBIR grant, Cellecta already had data showing that its sgRNA modifications could help improve knockout efficiency.
The specific changes the company made was to swap in an adenine in place of a thymine and to insert a five nucleotide extension into a stem-loop structure that interacts with the Cas9 enzyme.
The A-T switch breaks up a stretch of four thymines just a few nucleotides downstream of the approximately 20-nucleotide programmable part of an sgRNA. "That's a weak termination site," Tedesco said. Because sgRNAs are often delivered on a plasmid and transcribed using a promoter by RNA polymerase 3, "keeping those four Ts means not all the sgRNAs that started to be transcribed are going to make a full-length guide. A fraction are just going to terminate there." He added that five thymines denote a strong termination site.
Tedesco said that the termination signal jumped out to Cellecta's scientists and a literature review confirmed that others had noticed it as a potential problem. Similarly, when Emmanuelle Charpentier and colleagues created the first sgRNA, they didn't use the full complementary region of the crispr RNA and the tracr RNA. "They used just a part and they saw it works. What we saw is that maybe, they cut too much," he said.
Cellecta's researchers first tried targeting green fluorescent protein with the modified sgRNAs and saw an improvement, but wanted to see if the changes would help them better target endogenous genes. To do so, they created several libraries of 4,000 sgRNAs targeting multiple genes, including some known to be essential to cell function as positive controls, and a few that targeted introns as negative controls. The four libraries consisted of "wild type" sgRNAs, the A-T switch, the five nucleotide insertion, and a combo that featured both modifications.
The screens revealed several new genes that reduced cell viability. Moreover, across the genome, the modified sgRNAs outperformed the "wild type" counterparts.
"The A-T switch is what gives the biggest results. It's the main improvement," Tedesco said. And the combo proved to be slightly better than the A-T switch alone.
Having already incorporated the changes into the libraries it sells to pharmaceutical and biotech firms, Cellecta just recently submitted an application for a Phase II grant to further improve knockout efficiency.
For Phase II, the firm won't be making more changes to the sgRNA structure. Instead, it will try to design sgRNAs to target regions of genes that code for critical amino acids in proteins.
"The problem with the knockout efficiency with CRISPR is that cutting DNA is not enough," Tedesco said. "What knocks out the gene is the DNA repair mechanism trying to repair the DNA and then making mistakes. That doesn't have anything to do with the ability of cas9. You can cut out a sequence a million times, but if the DNA repair fixes it right, nothing changes."
The non-homologous end joining repair pathway often introduces short indels, but if those indels are any multiple of 3 nucleotides, it will simply add amino acids, which may still result in a functional protein.
"Even the best sgRNA you can design, on average, is going to introduce out-of-frame indels on both alleles only 50 percent of the time," Tedesco said. But theoretically, one could increase the chance of a total knockout by targeting protein domains known to be essential.
"If you are making sgRNAs for regions that overlap with catalytic sites of an enzyme, it's very likely a modification will cause a non-functional enzyme," he said.
Cellecta is proposing to develop a method to design genome-wide libraries that specifically target known functional domains. Part of the information needed to do so is already out there in proteomics databases and the literature.
"You just need good bioinformatics people that can map these protein regions onto genomics regions so you can design sgRNAs against them," he said. Cellecta will also take an evolutionary approach, comparing homologs in different species. "Any amino acid sequence that is more or less conserved across species, we know it was conserved by evolution, so it's likely important."
Tedesco said the firm expects to hear back from the NIH about the Phase II grant in three to six months.
These changes could help Cellecta improve the pre-made and custom screens it sells to pharmaceutical firms to identify new targets for therapies.
While the Phase II grant won't be incorporating any new structural changes to sgRNAs, Tedesco said that doesn't mean there aren't any still out there to be discovered.
"I don't see why there couldn't be other changes," he said. "We're still talking about [finding those]. In this screen, we just focused on a certain set of modifications, based on known sgRNA structure. What could be done is to do a deep mutagenesis screen on the constant structure and find other mutational insertions that increase the affinity of Cas9.
"We can't say we've hit the ceiling. We still don't see 100 percent knockout efficiency," he said.