NEW YORK (GenomeWeb) – Using molecular biology techniques, a scientist from the Italian Foundation for Cancer Research's Institute of Molecular Oncology (IFOM) has found a way to turn expressed mRNA into guide RNAs for CRISPR/Cas9 knockout screens.
"This method does not rely on bioinformatics and opens a path for forward genetics screening of any species, independent of their genetic characterization," Hiroshi Arakawa, the study's sole author, wrote in a paper published today in Science Advances.
His method extracts approximately 20 nucleotides from mRNA and incorporates them into CRISPR/Cas9 gRNA constructs using restriction enzymes and ligations in a multi-step process. But to bind DNA, that section of a gRNA must target a genomic region next to a three base pair segment called a protospacer-adjacent motif, which is "NGG" for the gold-standard CRISPR/Cas9 from Streptococcus pyogenes (SpCas9).
Arakawa found the appropriate PAM-adjacent 20 nucleotide sections in mRNA by using primers with a section complementary to the "NGG" PAM in them to synthesize cDNA. About a dozen steps later, the method yields a guide sequence DNA fragment that can be cloned into the gRNA scaffold in a vector for delivery into cells, where the gRNA can be expressed off the plasmid.
Arakawa, who studies hyper-targeted integration and gene conversion using chicken B cells, told GenomeWeb he was interested in genetic screening for his area of interest. "Genetic information is not very [well] characterized in chicken and there are no gRNA libraries, but I wanted to have one," he said. "Some cell type- or species-specific biological properties may be driven by uncharacterized or unannotated genes. For example, I suspect that these unknown genes may play a key role in [immunoglobulin] gene conversion or hyper-targeted integration in chicken B cells."
It took about three months to conceive the method to disrupt those genes, he said, and another five months to get it to work, testing a handful of different approaches for each of about a dozen steps: cDNA synthesis; a 3' linker ligation; Eco P151 digestion; a 5' linker ligation and Bgl II digestion; a PCR optimization; Acu I/Xba I digestion; another 3' linker ligation; a second PCR optimization; BSM BI/Aat II digestion; and finally cloning into the vector.
In his paper, Arakawa generated a gRNA library from the B cell transcriptome, limiting it in two ways: it was not genome-wide and rare transcripts were less likely to yield gRNAs.
But, on the plus side, he noted that the method generated multiple guide sequences for the same gene, an important feature of many CRISPR/Cas9 knockout screens, which often have several different gRNAs for each gene.
"It can be also useful to make personalized human gRNA libraries, which represent collections of single-nucleotide polymorphisms from different exons," Arakawa wrote. "These personalized human gRNA libraries could be used to study allelic variations and their phenotypes, leading to better characterizations of rare diseases."
Arakawa said he has applied for a patent on the method and is looking for business partners.