Scientists at the Wellcome Trust Sanger Institute have developed a technique to create a comprehensive library of mutations across all genes in the mouse genome. This library can be used to examine the role of every gene in different cell types, the researchers say.
CRISPR technology uses the DNA-cutting enzyme Cas9, with the help of a guide RNA sequence, to find and modify genetic targets. Scientists engineer multiple new guide RNAs using standard molecular biology techniques. This makes for a much faster and efficient method to modify the genome of any cell type in any species, according to the Wellcome group.
The team, which published their results online (“Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library”) in Nature Biotechnology, found that 50 of 52 guide RNAs tested successfully cut both copies of the genes. The high success rate for these engineered guide RNAs seems to be consistent across many cell types, which led them to create a library of guide RNAs targeting every gene in the mouse genome.
“We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9,” wrote the investigators. “Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and four previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.”
“CRISPR technology is revolutionizing how we study the behavior of cells,” said Kosuke Yusa, Ph.D., lead author from the Wellcome Trust Sanger Institute. “We've developed a thorough library that can be used by other researchers to study the role of any gene. We can create a library of this type for any cell or any species.”
The team will now use the engineered library to create mutations in cancer cell lines. A major problem with cancer drugs is that often cells can quickly acquire resistance and reject the treatment. By screening for genes that have lost all function through mutation, the team can determine what genes are involved in acquiring resistance to cancer treatments and thus find a clinical target.