SANTA CRUZ — A Ph.D. graduate student in biomolecular engineering at UC Santa Cruz, with a background in computer science and mathematics, has created an innovative software program called CRISPRware, which makes the process of gene editing faster and easier for researchers, including those developing treatments for genetic conditions such as sickle cell disease or cystic fibrosis.
The CRISPRware software was presented in a recently published paper in “BMC Genomics,” authored by the software’s creator, Eric Malekos, along with fellow Molecular, Cell and Developmental Biology Department Ph.D. student Christy Montano and with the guidance and encouragement of professor Susan Carpenter of UCSC’s eponymous Carpenter Lab.
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“I am an immunologist by training,” said Carpenter. “I’ve been studying all the molecular pathways involved in driving inflammation with interest in infection, so acute inflammation, but also interest in chronic inflammation, as it relates to autoinflammatory diseases. I started my lab at UCSC dominantly focusing on the role that RNA plays in regulating these responses.”
RNA, or ribonucleic acid, is similar to DNA, but instead of the coiled double-strand structure, RNA generally has only one strand and slightly different building blocks. RNA has a few jobs in the body, including acting as a messenger between DNA and a structure called a ribosome, which synthesizes proteins, essentially telling the ribosomes which proteins to make. However, the RNA that Carpenter and those in her lab study does not serve as an instigator for protein synthesis, and is called long non-coding RNA.
“When I was doing my training, RNA sequencing became a hot new technique and we discovered all of these RNA genes that go up and down after inflammatory responses or infections, but none of them code for protein,” said Carpenter. “They are basically placed into a pot where they are called long non-coding RNAs and there are 36,000 of these genes that have been identified now in the human genome. They greatly outnumber what we’ve been studying, which are protein-coding genes, and we really know very little about what they do. So, my lab dominantly focuses on understanding how these long non-coding genes play roles in regulating our immune responses.”
To study the non-coding RNA, Carpenter and her lab use the gene-editing tool CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, to locate and edit specific gene sequences. The cutting mechanism in CRISPR is generally a protein called Cas9, which is guided to snip a certain point on the genome by an RNA sequence, called a guide RNA.
“The big thing with CRISPR is that it gives you a lot of power to target a particular section of the genome,” said Malekos. “In the guide RNA, you have about 20 nucleotides and you can make that so that it matches the 20-nucleotide sequence in the genome. That makes it close to being able to target, with specificity, any particular region of the genome because you don’t see that many repeated 20-nucleotide sequences, so 20 nucleotides gives you the sort of specificity to hit distinctly different parts of the genome.”
The trouble was that the tools available to researchers only featured guide RNA for the approximately 20,000 known protein-coding genes in the human genetic code, but not others. The CRISPRware software, however, can scan an entire genome at once and identify all possible guide RNAs for any region of the sequence.
“When we do research, we typically use a model of the human genome, which is really an agglomeration of about 20 individuals which were originally used for the human genome sequencing project, which is to say that the sequence we use is not representative of each individual, because we’re all different and genetically have variation,” said Malekos. “One interesting component of my work is that you can incorporate specific genetic variations among people to find CRISPR guides that will work in some cases and not in others.”
Malekos ran the CRISPRware software on the genomes of six model species that are often used by researchers in various fields: human, rat, mouse, zebrafish, fruit fly and a roundworm known as Caenorhabditis elegans. The CRISPRware software then generated comprehensive catalogs of guide RNAs for each species. The software’s outputs were then uploaded into the publicly accessible UCSC Genome Browser, which celebrates its 25th birthday this month.
“This is a super cool feature,” said Carpenter. “Saying you’re working on a fruit fly and you want to target a gene but you’ve never used CRISPR before. You could, within a day, figure out which guide would work for the gene of interest. It speeds up the ability to use CRISPR in these six model organisms.”
Looking ahead, Malekos sees CRISPRware being adapted to study the genomes of complex cancer cells and plant cells, which can have more than a thousand chromosomes compared to the 46 chromosomes in a human cell.
“Typically, the genomes of cancer cells are really funky,” said Malekos. “They don’t look anything like a normal human cell in terms of the genome. They’ll have extra chromosomes and chromosomes attaching in weird ways. CRISPRware is made to work with two sets of chromosomes now so future work could be being able to consider these more complex cancer genomes. Along those lines, plants can have a huge number of chromosomes and that’s not something that CRISPRware is designed for right now.”
However, Carpenter and Malekos, who is a National Institute of Health predoctoral fellow, are concerned about the future of their research and the research of others with the recent confusion surrounding cuts by the Trump administration to the National Institute of Health.
“Science funding is really important,” said Carpenter. “We are excited to talk about our research and we think that it’s important that people do it more and more, so we know what’s at stake and what people can lose.”