CRISPR Offers Unrestricted Opportunities to Edit Genes

October 2014

A new addition to the genetic engineer’s toolbox has surfaced as a hot topic. Called CRISPR, for Clustered Regularly Interspaced Short Palindromic Repeats, in nature this is the basis of an adaptive immune system in bacteria and archea. In the hands of molecular biologists, CRISPR has become the heart of a newfound ability to precisely edit DNA. 

Underscoring the topic’s currency, CRISPR work now underlies nine Research Fronts in Thomson Reuters Essential Science Indicators, a subset of the Web of Science.  Research Fronts, identified via automated analysis of citation patterns, self-organize when a foundational “core” of earlier papers is frequently cited together by later reports, forming a distinct area of related research. In seven of these CRISPR clusters, the core papers, on average, date from 2013 or later. Such a comparatively “young” core signals a fast-moving area undergoing rapid development.  

A SPACER ODYSSEY

In microbes, each CRISPR repetition is made up of a short sequence of base pairs followed by more or less the same sequence in reverse (the palindrome) and then by spacer DNA of around 30 base pairs. This spacer is derived from previous viral infections that inserted viral DNA into the host DNA. Several such repetitions are usually accompanied by a set of CRISPR-associated (Cas) proteins that break nucleic acids. The CRISPR sequence is transcribed into a long precursor RNA molecule, which is cut just upstream of each spacer to yield a collection of small CRISPR RNA (crRNA) molecules. The spacer recognizes complementary sequences in the genome of any invading virus. Cas proteins then latch onto the crRNA and break the viral nucleic acid, neutralizing the invasion. Fragments released by Cas-mediated breaks—which don’t necessarily need a crRNA guide to trigger them—are incorporated into the host DNA as new spacer elements, which are then passed on when the microbe divides. So the descendants of that individual have a memory of a specific viral infection that forms the basis of their adaptive immune response.

All of which, while great for bacteria and archaea, is not the stuff of hot topics or Research Fronts. That required turning the microbial systems, originally described in 2000 by a Spanish research team under Francisco Mojica (see adjoining table, paper #10) and named a couple of years later by Ruud Jansen’s group in the Netherlands (paper #4) into useful tools.

Selected Papers on CRISPR/Cas Systems and Genome Engineering

Listed by citations

Rank Paper Citations
1    L. Cong, et al., “Multiplex genome engineering using CRISPR/Cas systems, Science, 339(6121): 819-23, 2013. [6 U.S. and Chinese institutions] 540
P. Mali, et al., “RNA-guided human genome engineering via Cas9,” Science, 339(6121): 823-6, 2013. [Harvard U. Sch. Med., Boston, MA; Boston U., MA] 531
 M. Jinek, et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Science, 337(6096): 815-21, 2012. [Howard Hughes Med. Inst., U. Calif., Berkeley; U. Vienna, Austria; Umea U., Sweden] 383
4 R. Jansen, et al., “Identification of genes that are associated with DNA repeats in prokaryotes,” Molec. Microbio., 43(6): 1565-75, 2002. [U. Utrecht, Netherlands; Natl. Inst. Publ. Hlth. & Environ. Protect., Bilthoven, Netherlands] 306
5 W.Y. Hwang, et al., “Efficient genome editing in zebrafish using a CRISPR-Cas system,” Nature Biotech., 31(3): 227-9, 2013. [Massachusetts Gen. Hosp. Boston; Harvard U. Sch. Med., Boston, MA; Broad Inst.,  Cambridge, MA] 262
H.Y. Wang, et al., “One step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering,” Cell, 153(4): 910-8, 2013. [Whitehead Inst., Cambridge, MA; MIT, Cambridge, MA; Broad Inst. Cambridge, MA] 256
S.W. Cho, et al., “Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease,” Nature Biotech., 31(3): 230-2, 2013. [Seoul Natl. U., South Korea] 178
8 W.Y. Jiang, et al., “RNA-guided editing of bacterial genomes using CRISPR-Cas systems,” Nature Biotech., 31(3): 233-9, 2013. [Rockefeller U., New York, NY; Broad Inst., Cambridge, MA; MIT, Cambridge] 156
M. Jinek, et al., “RNA-programmed genome editing in human cells, eLife, 10.7554/eLife.00471, 2013.  [U. Calif., Berkeley] 134
10 F.J.M. Mojica, et al., “Biological significance of a family of regularly spaced repeats in the genomes of Archea, bacteria, and mitochondria,” Molec. Microbio., 36(1): 244-6, 2000. [U. Alicante, Spain; U. Miguel Hernandez, Alicante, Spain]  94
11 T. Wang, et al., “Genetic screens in human cells using the CRISPR-Cas9 system,” Science, 343(6166): 80-4, 3 January 2014. [MIT, Cambridge, MA; Whitehead Inst., Cambridge, MA; Harvard U. Sch. Med., Boston, MA] 64
12 O. Shalem, et al., “Genome-scale CRISPR-Cas9 knockout screening in human cells,” Science, 343(6166): 84-7, 3 January 2014. [Broad Inst., Cambridge, MA; MIT, Cambridge, MA; Harvard U. Sch. Med., Boston, MA] 58

Source: Thomson Reuters Web of Science

Jennifer Doudna, of the University of California Berkeley, paved the way. Her group worked out how one of the CRISPR-associated proteins, Cas9, creates double-stranded breaks in DNA when guided by two crRNA molecules (or a single chimera created from the two crRNAs, which makes life easier).  Doudna et al. mentioned “the potential to exploit the system for RNA-programmable genome editing” (paper #3). The point being that the cell does not sit idly by and leave broken DNA alone; it tries to repair it, offering the genetic engineer opportunities to have the repair done to her specifications. Doudna’s team quickly followed up by demonstrating that they could construct guide RNAs analogous to CRISPR sequences that would target Cas9 to break the DNA at specific points along the DNA in human cells (paper #9). That effectively opened the floodgates to papers extending the system.

Most prominent among these are two papers published together in Science in early 2013, which top the citation counts for all the core papers in these Research Fronts, by virtue of showing the huge potential of CRISPR/Cas for both understanding and manipulating gene function. A large group led by Feng Zhang of the Broad Institute of MIT and Harvard created a system that used synthetic guide RNA and Cas9 to break DNA at precise sites in mouse and human cells. They also converted Cas9 into a nicking enzyme that breaks only one strand of the DNA, paving the way to precise gene repair. And by stringing different target spacer sequences together in a single CRISPR construct they simultaneously edited many sites in the human genome, what they called “multiplex genome engineering” (#1). In addition to multiplex engineering, George Church’s group at Harvard University School of Medicine, used CRISPR/Cas on human cells to repair an inactivated gene and to insert a selectable marker directly into the DNA at a chosen point (#2)

RAPID DEVELOPMENT

One of the most remarkable aspects of the development of CRISPR/Cas is how rapidly it took place, thanks largely to its relative simplicity. As Jennifer Doudna said in an interview for BioTechniques (54: 185-8, April 2013), “it’s a really great example of how fundamental basic research, which was not aimed at any particular target or goal or certainly a particular application, led to the discovery of a system that may turn out to be a really transformative technology for genome engineering.”

That can be seen in the rapid broadening of the scope of CRISPR/Cas. In the space of less than a year, Rudolf Jaenisch at the Whitehead Institute for Biomedical Research in Cambridge, MA, and his group generated mice with several target genes rendered non-functional (#6). Previously, creating a mouse with several mutated genes required either knocking out one gene at a time in embryo stem cells, with low success rates at each stage, or else cross-breeding several types of mice, each with a single mutation, which is both time-consuming and inefficient. Jaenisch’s group knocked out five genes at once in embryo stem cells and used CRISPR/Cas in fertilized eggs to engineer adult mice with precise point mutations in two separate genes.

David Bikard at Rockefeller University, working with Feng Zhang, used modified CRISPR sequences to target specific genes in bacteria and obtained very efficient editing of their target genes. They also figured out ways to target difficult sequences, further extending the value of the technique (#8)

A Korean team under Jin-Soo Kim of Seoul National University used Cas9 and synthetic guide RNAs to knock out a couple of genes in human cells (#7) while J. Keith Joung at Massachusetts General Hospital and his group extended the system to zebrafish and knocked out eight genes in a single shot (#5) The method was also quickly applied to plants, both model species such as Arabidopsis and important crops such as rice (K. Belhaj, et al., Plant Methods, 9: No. 39, 2013: 11 citations in Web of Science at this writing)

More recently, two groups have shown the power of CRISPR/Cas to undertake genome-wide screens, using tens of thousands of guide RNAs to target up to 20,000 genes across the entire genome, uncovering, for example, some new candidates for genes that confer resistance to anti-cancer drugs (#11) and (#12). A Chinese team used CRISPR/Cas in fertilized eggs to create monkeys carrying two specific mutations, never before achieved in primates (Y.Y Niu, et al., Cell, 156[4]: 836-43, 13 February 2014; 24 citations). And a group led by Daniel Anderson at MIT successfully corrected a mutation in adult mice that model the human disease hereditary tyrosinemia (H. Yin, et al.,  Nature Biotechnology, 32(6): 551-3, June 2014; 10 citations).

ANOTHER TRANSFORMATIVE TOOL

The excitement is thus very well deserved. For a biologist, though, one of the most interesting aspects of the CRISPR/Cas story is the way that it mirrors an earlier transformative tool: restriction enzymes. As first investigated by Salvador Luria in the early 1950s, restriction was a troublesome phenomenon whereby bacteriophages wouldn't grow well on some strains of bacteria. It turned out that the bacteria had enzymes that recognized viral sequences and cut the phage DNA, restricting the growth of the virus. Fast forward almost 20 years and the innate immune system of hundreds of species of bacteria supplied genetic engineers with a well-stocked toolbox of precision scissors—restriction enzymes—that enabled them to snip DNA almost anywhere they chose, and thus to manipulate DNA with unprecedented precision. In similar fashion, with somewhat less delay between discovery and application, CRISPR/Cas represents a new class of restriction enzymes that gives microbes an adaptive immune system and hands genetic engineers another wonderfully capable new set of tools.


Dr. Jeremy Cherfas is a science writer based in Rome, Italy.

The data and citation records included in this report are from Thomson Reuters Web of ScienceTM. Web of ScienceTM is a registered trademark of Thomson Reuters. All rights reserved.