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CRISPR: A New Tool For Gene Editors

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CRISPR Gene Editing—Poised to Revolutionize Neuroscience?

Bacteria have been doing it for millennia. Researchers, on the other hand, have only recently learned how to wield CRISPR—a gene-editing tool that bestows the power to delete, add, or toy with the expression of genes at virtually any site in the genome. The magic works in every creature tested, from plants to primates. CRISPR’s flashy reputation has prompted some doubts, such as reported off-target effects, but those are gradually being laid to rest by so-called “CRISPR jocks.”

By now, neuroscientists are on the scent. Two recent studies reported that the method effectively silences genes within neurons, both in hippocampal slice cultures and within the brains of living mice. CRISPR promises to dramatically speed up the generation of transgenic animals. It also may allow researchers to repair mutations in neural stem cells, and possibly facilitate gene therapy in humans. Researchers have already corrected disease-causing mutations in mouse models of muscular dystrophy and a fatal liver disease. CRISPR studies of neurodegenerative diseases are just starting to ramp up, as researchers are using the new technique to build animal models and develop potential therapies.

“CRISPR is a game-changer, and it’s utterly simple to use,” Roger Nicoll of the University of California, San Francisco, told Alzforum. An electrophysiologist who claims to know next to nothing about manipulating genes, Nicoll used CRISPR to delete glutamate receptors in hippocampal neurons, as reported in the September 3 Neuron. “It was the most satisfying, uncomplicated study that I’ve done,” he said.

CRISPR Kaleidoscope. Cas9 nuclease unfurls itself to bring a guide RNA and its target DNA sequence together. Once bound, the enzyme makes a double-stranded DNA break, initiating CRISPR-mediated genome editing. 

The acronym CRISPR, for clustered regularly interspaced short palindromic repeats, was first coined in 2002 to describe a phenomenon uncovered by decades of research. Nearly half of all bacterial species and almost all archaeal species whose genomes have been sequenced harbor a curious breed of clustered repeat sequences. These repeats were interspersed between nonrepetitive sequences, which later proved to be snippets of genetic material from phages, the viruses that infect bacteria (see Jansen et al., 2002). Eventually, researchers figured out that bacteria had used these nonrepetitive sequences as a defense against phage infection. When phages invade, the bacteria transcribe these captured DNA sequences, at which point a bacterial nuclease called Cas9 dices up the transcripts and forms complexes with the pieces. Then the RNA fragments pair up with complementary sequences in the invading phage DNA, whereupon Cas9 exacts a double strand break. As these genomic fractures grow in numbers, they ultimately neutralize the phage.

Phage Hitman. Researchers first discovered CRISPR as a bacterial defense system against phage viruses. The Cas9 nuclease snips phage DNA and pockets it in the bacterial genome as novel spacer sequences. The nuclease later transcribes those to hunt down complementary sequences in the phage and dice up its DNA. [Courtesy of James Atmos, Wikimedia Commons.]

Researchers next learned that they could co-opt this system to silence any gene. After double strand breaks occur, the host naturally repairs them using a process called non-homologous end joining. NHEJ is a haphazard type of repair that adds or removes nucleotides to the target sequence to ligate the broken ends, usually causing a frameshift that renders the gene defunct. 

Just last year, scientists got the system to work in mammalian cells by transfecting them with appropriate guide RNAs along with the Cas9 gene derived from Streptococcus pyogenes (see Cong et al., 2013, and Mali et al., 2013). Since then, applications have evolved at a blistering pace. Researchers can now use CRISPR to edit or add genes. After transfecting cells with desired “filler” sequences flanked with DNA homologous to the insertion site, cells are coaxed to use homologous recombination, rather than NHEJ, to insert the foreign fillers into the host DNA. Using this more controlled technique, researchers can correct mutations or add new ones, insert reporter sequences, or knock in entire genes. Some researchers even fiddle with gene expression by using the CRISPR complex as a delivery vehicle for transcriptional activators and repressors, rather than as a DNA editor (see Gilbert et al., 2013, and Maeder et al., 2013). The technology eventually will supplant popular gene-editing technologies based on zinc finger and TALEN endonucleases, which are more time-consuming and costly to run than CRISPR, scientists believe. “Right now, it seems that there are no limits to what you can use [CRISPR] for,” Nicoll said.

Nicoll jumped aboard the CRISPR bandwagon to knock out glutamate receptors in neurons within hippocampal slice cultures. His lab had previously used the Cre/Lox system to do this. “The limitation there is, you have to make the whole mouse,” he said. This process can take years if more than one mutation or transgene is desired. “Now CRISPR comes along, and you can make multiple mutations simultaneously in about a month or two, the time it takes you to make the constructs,” Nicoll said.

Co-first authors Salvatore Incontro and Cedric Asensio cut their teeth on CRISPR by targeting the subunits GluN1 and GluA2 of the NMDAR and AMPAR glutamate receptors that are expressed in excitatory synapses. (These are among the receptors that have been proposed to bind Aβ oligomers and mediate neurotoxic consequences in Alzheimer’s disease.) The researchers coated plasmids containing complementary guide GluN1 and GluA2 RNAs, along with a plasmid containing Cas9, onto gold particles, and blasted them onto hippocampal slice cultures using a gene gun. Every transfected neuron they looked at failed to produce excitatory signals when given the proper stimulus, suggesting that their glutamate receptors were gone. The phenotypes were identical to those seen in Cre/Lox-based subunit knockouts.

Another paper, published in PLOS One a day before Nicoll’s, also reported knocking down the same GluN1 subunit in neuronal slice cultures. Led by Bernardo Sabatini at Harvard University, this study used CRISPR in utero. First author Christoph Straub and colleagues injected the CRISPR constructs directly into the brains of mouse embryos 15 days after conception—a time when neuronal progenitors are starting to differentiate into bona fide neurons, Sabatini told Alzforum. Two to three weeks after the pups were born, the researchers measured recordings of hippocampal neurons, and found that every transfected cell was devoid of NMDAR-mediated signals. The in utero CRISPR technique allowed the researchers to knock down a gene in cells within their native developmental context, Sabatini said. He added that the technique offered a refreshing departure from issues known to plague RNA interference experiments, such as incomplete knock down. “We, as a community, are tremendously excited about using CRISPR in neurons, mostly because we have had so much trouble with RNAi,” Sabatini said. “I have no doubt that any lab that has ever done RNAi is trying this.”

To learn how scientists use CRISPR to make transgenic animals, and about early forays into neurodegenerative disease research, read part 2 of this story.—Jessica Shugart

 

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References

News Citations

  1. Neuroscientists Probe CRISPR Transgenics and Treatment Paradigms

Paper Citations

  1. Jansen R, Embden JD, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002 Mar;43(6):1565-75. PubMed.
  2. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013 Feb 15;339(6121):819-23. Epub 2013 Jan 3 PubMed.
  3. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science. 2013 Feb 15;339(6121):823-6. Epub 2013 Jan 3 PubMed.
  4. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013 Jul 18;154(2):442-51. Epub 2013 Jul 11 PubMed.
  5. Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK. CRISPR RNA-guided activation of endogenous human genes. Nat Methods. 2013 Oct;10(10):977-9. Epub 2013 Jul 25 PubMed.

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Neuroscientists Probe CRISPR Transgenics and Treatment Paradigms

CRISPR, a gene-editing technology borrowed from humble bacteria, has taken the biomedical research world by storm. Researchers can use the technique to inactivate genes, add point mutations, or insert entire genes into virtually any spot in the genome of any species. Neuroscientists are just starting to dabble with CRIPSR’s gene-editing recipe, and the first studies are cropping up (see part 1). CRISPR promises to speed up production of transgenic animals, reverse fatal diseases in animal models, and maybe one day even correct disease-causing mutations in people.

Transgenic Animals Made Easy
In addition to manipulating cell and animal genomes, another major advance CRISPR offers is the rapid generation of transgenic animals. Such models have been the cornerstone for much of the basic and translational research in the neurodegenerative disease field, but they require an extraordinary amount of time and resources to make. The tried-and-true way to generate transgenic mice involves transfecting embryonic stem cells with the chosen DNA, selecting transfected cells, injecting them into mouse blastocysts, and eventually selecting for offspring in which the transgene jumps into the germline. In contrast, simple direct injection of Cas9 plus multiple guide RNAs into mouse embryos has allowed researchers to skip several of these steps and produce multi-knockout mice in one fell swoop.

Mike Sasner of The Jackson Laboratory in Bar Harbor, Maine, expressed enthusiasm about CRISPR’s ability to accelerate development of transgenic mice. “It’s trimming the first part of the pipeline significantly, in terms of time and cost, and that means more mice can be made."

In a recent study, researchers led by Rudolf Jaenisch at the Massachusetts Institute of Technology used the technique to disrupt up to five genes—Tet1, 2, and 3, and the Sry and Ury genes. The Tet genes are involved in the production of 5-hydroxymethylcytosine, and knocking down more than one of them at once allowed researchers to avoid the pitfall of redundancy that frequently undermines single-gene knock-out models (see Wang et al., 2014). 

Benedikt Wefers of the German Center for Neurodegenerative Diseases (DZNE) in Munich is using CRISPR to make mice with AD-associated point mutations, as well as knock-ins of human disease-related genes. The homology-dependent process of adding a specific mutation into a mouse gene or a whole human gene is less efficient than using NHEJ to inactivate genes, Wefers said. Hence he found that most of the initial offspring harbored inactivated genes rather than added ones. “If you are interested in a knock-in or point mutations, you have to screen more mice than you do for knockouts,” he said. Wefers, who directs DZNE’s transgenic-mouse unit, estimated that the homology-directed repair works about 5 percent of the time, so the researchers typically screen 30 to 60 pups to find appropriate founders. Still, he said the process is far more streamlined than creating mice by traditional techniques based on embryonic stem cells.

Other transgenic animals could be generated in the same way. The creation of transgenic mammals has, for the most part, been limited to mice, JAX’s Sasner added, because embryonic stem cell technology does not readily work in other species. With this step removed, researchers could theoretically generate a menagerie of transgenics. “CRISPR can work in any species,” Sasner said. “It is a huge step forward.” Researchers have already used the technique to make transgenic monkeys with simultaneous alterations in the recombination activating gene 1 (Rag1) and peroxisome proliferator-activated receptor gamma (PPAR-γ) genes, which are crucial for adaptive immunity and metabolism, respectively (see Niu et al., 2014). 

Zebrafish have proven to be easy targets for CRISPR, according to Bettina Schmid, also at DZNE. “We are absolutely fascinated by the CRISPR/Cas9 system. It allows us to do experiments we only were dreaming about years ago,” she wrote in an email to Alzforum. “It is easy, cheap, everybody can do it, and it is efficient.” Her lab started off using CRISPR to inactivate genes in zebrafish, and is now introducing point mutations associated with neurodegenerative disease.

Sasner said that he expects he will soon see CRISPR-built transgenic mouse models of neurodegenerative disease, although none have yet been submitted to the Jackson Lab. This not-for-profit organization maintains, and supplies researchers with more than 7,500 well-characterized mouse strains, including many Alzheimer’s models. “In 2015, I think we are going to see a flood of mice created with this technology,” he said. Sasner added that CRISPR will allow mouse modelers to keep closer pace with the discovery of new disease-associated genes and genetic variants found in massive genome-wide association studies. Rather than rolling the dice by targeting a single gene with a single mutation, researchers can more readily make multiple mice that model several different GWAS hits.

Todd Golde at the University of Florida in Gainesville agrees. “It’s going to be very hard to use traditional means to find out how all of these novel [GWAS] genes supposedly influence disease,” he said. “CRISPR gives us a faster, more facile tool to do that.”

Rather than generating transgenic animals from the embryo stage, Golde plans to use CRISPR to inactivate genes in mice after they are born. In collaboration with Edgardo Rodriguez, a new investigator at the University of Florida who works on CRISPR delivery systems, Golde plans to use adeno-associated virus (AAV) vectors to target CRISPR directly to the central nervous system (CNS). The scientists plan to inactivate genes while avoiding potential pitfalls associated with transgenic mice, such as developmental defects and uncertainty about the influence of genetic background, he said. While Golde hopes eventually to examine the effects of novel disease genes, his lab will begin their venture into CRISPR territory by looking at the effects of knocking down familiar culprits, such as BACE1 and APP.

Beyond mice, CRISPR will allow researchers to rapidly model a slew of genetic mutations in human cells. For example, researchers will be able to easily introduce, or correct, mutations in induced pluripotent stem cells (iPSCs) derived from patients, and then study the effects of those manipulations in a variety of differentiated cell types, including neurons.

“Anyone studying iPS cells would be crazy not to pursue this technology,” said Mathew Blurton-Jones of the University of California, Irvine. Blurton-Jones plans to use CRISPR to compare the effects of disease-associated mutations in the TREM2 gene, a known risk factor for AD. He will study the effects of either correcting the mutations in iPSCs derived from AD patients, or introducing them into iPSC lines from normal controls. Differentiating these cells into microglia, which express TREM2, will allow him to model disease and hunt down pathogenic mechanisms.

CRISPR for Therapy
Beyond basic and translational research, will CRISPR ever work as gene therapy? The answer to this question isn’t yet in, but some researchers are proposing that an early therapeutic application could be to correct mutations or add therapeutic genes to iPSCs before transplanting them back into the donor (see Li et al., 2014, and Kim et al., 2014). The hope is that such cells may treat diseases including amyotrophic lateral sclerosis, Parkinson’s, and perhaps even Alzheimer’s (see Sep 2013 news story, Sep 2010 news story).

Along those lines, Blurton-Jones plans to use CRISPR to insert potentially therapeutic genes—such as the growth factor BDNF or the Aβ-degrading enzyme neprilysin—directly into iPSCs. In a recent study, he reduced pathology in AD mouse models by injecting them with iPSC-derived neural precursors overexpressing neprilysin (see Blurton-Jones et al., 2014). However, he inserted that transgene randomly into the cell genome. “That’s fine for a basic science experiment, but not for the clinic,” Blurton-Jones said, adding, “We would need to target a specific locus that we know is safe, and CRISPR dramatically increases our ability to do that."

There are early hints that CRISPR could even be used to wipe out genetic diseases. A study published in Science Express on August 14 reported preventing Duchenne muscular dystrophy in mice through CRISPR repair of a single disease-causing genetic variation (see Long et al., 2014). Led by Eric Olson at Univeristy of Texas Southwestern in Dallas, first author Chengzu Long and colleagues corrected a point mutation that creates a stop codon in the dystrophin gene, which is crucial for muscle function. They injected mouse embryos with DNA encoding Cas9, guide RNAs for the mutated gene, and a wild-type replacement fragment. Dystrophin expression was restored in the resulting pups, and their muscle fibers never degenerated as the mice grew. Olson told Alzforum that his lab is now pursuing the next step toward a therapy, i.e., to reverse causative mutations in adult mice.

In another example, earlier this summer researchers led by Daniel Anderson at the Massachusetts Institute of Technology used CRISPR to rescue adult mice from hereditary tyrosinemia type I (HTI), a fatal liver disease caused by a point mutation in the fumarylacetoacetate hydrolase (FAH) gene (see Yin et al., 2014). The researchers delivered CRISPR/Cas9 into the mice’s liver via hydrodynamic injection, a method that uses large volumes of fluid to force nucleic acids into target cells. This delivery method causes some cellular damage and is unlikely to be applied to humans.

The major obstacle that stands in the way of CRISPR as a therapy for neurodegenerative disease is the same as for other potential gene therapies, that is, crossing the blood brain barrier (BBB) to deliver the genetic cargo to target cells. Scientists have tried to overcome it by using the adeno-associated virus AAV9, which crosses the BBB and infects neurons and astrocytes. Alas, AAV9 is too small to readily carry the Cas9 gene plus the other components that would be needed for CRISPR. Some researchers are trying to pare the Cas9 sequence back to its bare essentials to squeeze it into AAV9, while others propose using multiple viruses containing different parts of the gene that splice together when expressed in the same cell. A study in the September 4 Biotechnology Journal reported generating an “AAV toolbox” for CRISPR, which managed to pack the Cas9 and guide RNA sequences into one vector (see Senis et al., 2014). The researchers used this streamlined vector to target liver cells in mice.

The University of Florida’s Rodriguez is optimizing the AAV system to deliver CRISPR to the CNS. While he prefers to keep the details of his upcoming study under wraps, he hinted to Alzforum that the Cas9 protein derived from S. pyogenes represents but one form of the nuclease, and those from other bacteria could potentially work better with AAV or perform different functions. “There is an immense number of bacterial species that one can tap to develop these systems, so we have an enormous chest of tools,” he said.

Casey Maguire of Harvard University, who develops systems for delivering gene therapies into the brain, thinks AAV’s capacity may be the least of the problems. “When people want to fit something into AAV, they usually find a way to do it,” he said. But once the CRISPR system is fashioned to fit into AAV, other hurdles may lie ahead, he predicted. These include immune responses to the bacterial Cas9 protein and sustained expression after AAV infection. “Long-term expression is great if you want to overexpress a gene,” Maguire said. “But with a [gene] correction system, you want to correct it and get the heck out of there.” Maguire’s concern stems from the potential for off-target effects of the Cas9 nuclease, which has been shown to latch onto the wrong sequences in some cell culture studies. Theoretically this could lead to unpredictable disruptions in essential genes, although studies in mice and monkeys have not shown evidence of this effect so far. Some claim that off-target effects are less of a concern in vivo, where lower amounts of CRISPR components are transfected per cell. Researchers are also rapidly developing tricks to maximize CRISPR’s specificity (see Fu et al., 2014; Veres et al., 2014; and Tsai et al., 2014). 

Maguire suggested non-viral delivery systems such as exosomes as one strategy to avoid the risks of long-term expression. Exosomes are membrane-bound vesicles that are shed from cells and can be engulfed by other cells. Because researchers could package them with therapeutic nucleic acids and proteins, exosomes derived from a patient’s own cells could become a promising delivery system, Maguire said.

Keith Joung of Massachusetts General Hospital in Charlestown has for the past decade worked on gene editing and tweaked CRISPR to boost its specificity. He told Alzforum that the sheer amount of enthusiasm and effort researchers are now bringing to this technology should propel it forward rapidly. “It’s a very exciting time. I think the technology really is transformative, both in terms of what it can do for the research community and in terms of therapeutics.”—Jessica Shugart

 

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References

News Citations

  1. CRISPR Gene Editing—Poised to Revolutionize Neuroscience?
  2. Self-Derived Stem Cells Fly Under Monkey’s Immune Radar
  3. Where in the World Are the iPS Cells?

Paper Citations

  1. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013 May 9;153(4):910-8. Epub 2013 May 2 PubMed.
  2. Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, Guo X, Bi Y, Si C, Hu B, Dong G, Wang H, Zhou Z, Li T, Tan T, Pu X, Wang F, Ji S, Zhou Q, Huang X, Ji W, Sha J. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell. 2014 Feb 13;156(4):836-43. Epub 2014 Jan 30 PubMed.
  3. Li M, Suzuki K, Kim NY, Liu GH, Izpisua Belmonte JC. A cut above the rest: targeted genome editing technologies in human pluripotent stem cells. J Biol Chem. 2014 Feb 21;289(8):4594-9. Epub 2013 Dec 20 PubMed.
  4. Kim HS, Bernitz J, Lee DF, Lemischka IR. Genomic editing tools to model human diseases with isogenic pluripotent stem cells. Stem Cells Dev. 2014 Jul 30; PubMed.
  5. Blurton-Jones M, Spencer B, Michael S, Castello NA, Agazaryan AA, Davis JL, Müller FJ, Loring JF, Masliah E, LaFerla FM. Neural stem cells genetically-modified to express neprilysin reduce pathology in Alzheimer transgenic models. Stem Cell Res Ther. 2014 Apr 16;5(2):46. PubMed. Correction.
  6. Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson EN. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science. 2014 Sep 5;345(6201):1184-8. Epub 2014 Aug 14 PubMed.
  7. Yin H, Xue W, Chen S, Bogorad RL, Benedetti E, Grompe M, Koteliansky V, Sharp PA, Jacks T, Anderson DG. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol. 2014 Jun;32(6):551-3. Epub 2014 Mar 30 PubMed.
  8. Senís E, Fatouros C, Große S, Wiedtke E, Niopek D, Mueller AK, Börner K, Grimm D. CRISPR/Cas9-mediated genome engineering: An adeno-associated viral (AAV) vector toolbox. Biotechnol J. 2014 Sep 4; PubMed.
  9. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 2014 Mar;32(3):279-84. Epub 2014 Jan 26 PubMed.
  10. Veres A, Gosis BS, Ding Q, Collins R, Ragavendran A, Brand H, Erdin S, Talkowski ME, Musunuru K. Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell. 2014 Jul 3;15(1):27-30. PubMed.
  11. Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol. 2014 Jun;32(6):569-76. Epub 2014 Apr 25 PubMed.

External Citations

  1. Alzheimer’s models

Further Reading

Papers

  1. Maguire CA, Ramirez SH, Merkel SF, Sena-Esteves M, Breakefield XO. Gene Therapy for the Nervous System: Challenges and New Strategies. Neurotherapeutics. 2014 Aug 27; PubMed.
  2. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014 Jun 5;157(6):1262-78. PubMed.
  3. Pennisi E. The CRISPR craze. Science. 2013 Aug 23;341(6148):833-6. PubMed.
  4. Horii T, Arai Y, Yamazaki M, Morita S, Kimura M, Itoh M, Abe Y, Hatada I. Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering. Sci Rep. 2014 Mar 28;4:4513. PubMed.