NEW YORK (GenomeWeb) – Scientists from the University of California, Berkeley have developed a new way to efficiently create genome-wide libraries of guide RNAs (gRNAs) for CRISPR-based studies. The libraries can be used both for gene editing knock-out screens as well fluorescent tagging of chromosomes.
Led by Andrew Lane and Rebecca Heald, the scientists developed a method to create gRNAs from the genome of an organism itself, rather than making them synthetically. "We reasoned that potentially any DNA sequence could be enzymatically processed into a library of sgRNAs and used to tile along a chromosomal region," the authors wrote in the study published today online in Developmental Cell.
The gRNA is what makes CRISPR/Cas9 such a flexible and powerful technology, able to target almost any part of the genome. The programmable part of a standard gRNA is 20 nucleotides long and must be complementary to a site immediately 5' to an NGG triplet, known as a protospacer adjacent motif (PAM). These complementary sections are part of the longer single-stranded gRNA.
To create the libraries, the researchers used restriction enzymes to cut out 20 to 21 nucleotide-long DNA fragments proximate to PAM sites throughout the genome. They then transcribed those fragments to RNA and linked them with a 93-nucleotide 3' gRNA Cas9 hairpin to create full-length gRNAs.
The scientists named the method CRISPR-EATING, the latter part short for "Everything Available Turned into New Guides."
CRISPR-EATING has a few limitations. "The precise composition of a guide library cannot be defined as explicitly as it could be in a synthetic oligonucleotide-based library, raising the possibility that individual guides within the library may target more than one location in the genome," the authors wrote.
But the paper demonstrated two applications for this enzymatic method of creating gRNAs: fluorescently labeling chromosomes and genome-wide knockout screens.
To tag chromosomes, Lane and his colleagues didn't actually use the whole genome, he told GenomeWeb. Instead, they used a computational tool to analyze which sections of the genome would produce the most effective guides.
Lane did, however, use the whole genome to create libraries for knock-out screens of Escherichia coli. Creating a library of gRNAs for that bacterium cost less than $100, and Lane suggested that CRISPR-EATING could enable big savings compared to synthetic methods of creating gRNAs.
"We can make these libraries for a lot less money, which makes genetic screening potentially accessible in organisms less well studied," even those organisms whose genomes have not yet been sequenced, he said.