It is often desirable to generate recessive loss-of-function mutations in emergent model organisms; however, identifying such mutations in the heterozygous condition is challenging. Taking advantage of the CRISPR
/Cas9 genome-editing method (1
), we have developed a strategy to convert a Drosophila heterozygous recessive mutation into a homozygous condition manifesting a mutant phenotype. We reasoned that autocatalytic insertional mutants could be generated with a construct having three components: (i) A Cas9 gene (expressed in both somatic and germline cells), (ii) a guide RNA
(gRNA) targeted to a genomic sequence of interest, and (iii) homology arms flanking the Cas9-gRNA cassettes that match the two genomic sequences immediately adjacent to either side of the target cut site (Fig. 1A
). In such a tripartite construct, Cas9 should cleave the genomic target at the site determined by the gRNA (Fig. 1A
) and then insert the Cas9-gRNA cassette into that locus via homology-directed repair (HDR
) (Fig. 1, B and C
). Cas9 and the gRNA produced from the insertion allele should then cleave the opposing allele (Fig. 1D
), followed by HDR
-driven propagation of the Cas9-gRNA cassette to the companion chromosome (Fig. 1, E and F
). We refer to this trans-acting mutagenesis scheme as a mutagenic chain reaction (MCR
Video. CRISPR: A word processor for editing the genome
Fig. 1. Scheme outlining the mutagenic chain reaction (MCR). (A to C) A plasmid consisting of a core cassette carrying a Cas9transgene, a gRNA targeting a genomic sequence of interest, and flanking homology arms corresponding to genomic sequences abutting the target cleavage site (A) inserts the core Cas9-gRNA cassette into the targeted locus via HDR [(B) and (C)]. (D to F) In turn, the inserted cassette expresses both Cas9 and the gRNA, leading to cleavage (D) and HDR-mediated insertion of the cassette into the second allele, thereby rendering the mutation homozygous [(E) and (F)]. HA1 and HA2 denote the two homology arms that directly flank the gRNA-directed cut site.
The CRISPR/Cas9 gene editing technique takes advantage of an immune process that was discovered in bacteria and applies it to any gene of interest. This YouTube video created by the McGovern Institute for Brain Research with the help of Feng Zhang, one of the leaders in developing this technique for use in research, beautifully animates how this technique is used to create homology-directed genome insertions. The authors of this paper used homology-directed repair to insert their mutagenic chain reaction construct into fly DNA.
Mutagenic chain reaction
The design of the MCR construct is simple and elegant and is shown in the bottom portion of Figure 1A. It expresses the Cas9 DNA sequence, which will produce the Cas9 protein when translated, and the guide RNA sequence that will target the endonuclease to the fly *yellow* locus.
A crucial part of the design is the addition of regions of DNA called homology arms. These regions match the DNA sequence in the fly genome where the authors want the construct to be inserted. When Cas9 cuts the fly DNA, the presence of the homology arms in the MCR construct allow the cell to use that sequence as a template to repair the damaged DNA, thus inserting the construct into the genome.
Creating MCR flies
To insert the construct into flies, the authors injected it into wildtype fly embryos. They also injected separate sources of Cas9 DNA and the gRNA. They needed to inject a separate source of Cas9 to perform the initial DNA cut so the MCR construct could be inserted into one copy of the gene of interest. This video shows a researcher using a micro pipette to inject fly embryos.
We expected that autocatalytic allelic conversion by MCR
should be very efficient in both somatic and germline precursor cells, given the high frequency and specificity of mutagenesis (3
) and efficacy of homology-based integration (4
) mediated by separate genome-encoded Cas9 and gRNA genes observed in previous studies. We tested this prediction in D. melanogaster with the use of a characterized efficient target sequence (y1) (5
) in the X-linked yellow (y) locus as the gRNA target and a vasa-Cas9 transgene as a source of Cas9 (Fig. 2C
) because it is expressed in both germline and somatic cells (4
). As the defining element of our MCR
scheme, we also included two homology arms, ~1 kb each, flanking the central elements (Fig. 2C
) that precisely abut the gRNA-directed cut site. Wild-type (y+) embryos were injected with the y-MCR
element (see supplementary materials), and emerging F0
flies were crossed to a y+ stock. According to Mendelian inheritance, all F1
female progeny of such a cross should have a y+ phenotype (i.e., F1
females inherit a y+ allele from their wild-type parent).
Fig. 2. Experimental demonstration of MCR in Drosophila. (A) Mendelian male inheritance of an X-linked trait. (B) Theoretical MCR-based inheritance results in the initially heterozygous allele converting the second allele, thereby generating homozygous female progeny. (C) Diagram of y-MCR construct. Two y locus homology arms flanking the vasa-Cas9and y-gRNA transgenes are indicated, as are the locations of the PCR primers used for analysis of the genomic insertion site (see supplementary materials). (D) PCR analysis of a y+ MCR-derived F2♂ (lanes 1 to 3; see fig. S1 for sequence), yMCR F1♀ (lanes 4 to 6), and yMCR F1♂ (lanes 7 to 9) showing junctional bands corresponding to y-MCR insertion into the chromosomal y locus (lanes 2, 3, 5, 6, 8, and 9) and the presence (lanes 1 and 4) or absence (lane 7) of a PCR band derived from the y locus. Although the yMCR F1♂ (carrying a single X chromosome) displays only MCR-derived PCR products (lanes 8 and 9), yMCR F1♀s generate both MCR and noninsertional amplification products. (E) Summary of F2 progeny obtained from crosses described in table S1. (F) Low-magnification view of F2 progeny flies from an yMCR × ♂ x y+♀ cross. Nearly all female progeny display a y– phenotype. (G) High-magnification view of a full-bodied yMCR F1♀. (H) A rare 50% left-right mosaic female. (I) A y+ control fly.
Panels A and B in this figure describe how we would expect an X-linked trait (an allele of a gene located on the X chromosome) to be passed from parent to offspring based on the laws of Mendelian genetics. In this case, a mutation in the father can only be passed to the female offspring, because the males must inherit the Y chromosome from their father. If only one parent has the mutation, the females will be heterozygous. In the case of MCR inheritance, all females who inherit the mutation from their father will be homozygous, because one chromosome causes a mutation on the other chromosome.
Panel E demonstrates that the authors did not observe the traditional X-linked inheritance pattern in the offspring of their fly crosses, but saw the expedited MCR inheritance! In the first row, the authors summarize the offspring from the cross depicted in panel B. With the exception of one brown female, all female offspring were yellow. Panel F shows an image of the fly offspring from this cross.
Genotyping the flies
Panel C diagrams how the MCR construct was inserted into the genomic y locus as well as the PCR primers the authors used to determine if the MCR construct was successfully inserted. In panel D, you can see different bands of DNA that represent either the wildtype y locus or the homology arms from the MCR construct. Lanes 1-3 indicate that the male fly did not have the insertion, because only the wildtype band is seen. Lanes 4-6 indicate that the female fly was mosaic for the MCR insertion, because all three bands are seen. Lanes 7-9 show a male fly that carries the MCR insertion.
Not completely perfect
The mutagenic chain reaction, though very powerful, is not always perfect. For reasons the authors don't completely understand, they occasionally observed chimeric female flies, like the one shown in Panel H. Half of this fly's body is yellow while the other half is brown.
From two independent F0
male (♂) × y+ female (♀) crosses and 7 F0
♀ × y+♂ crosses, we recovered y– F1
♀ progeny, which should not happen according to Mendelian inheritance of a recessive allele. Six such yMCR
♀ were crossed individually to y+♂, resulting in 95 to 100% (average = 97%) of their F2
progeny exhibiting a full-bodied y– phenotype (Fig. 2, E and G
, and table S1), in contrast to the expected rate of 50% (i.e., only in males). We similarly tested MCR
transmission via the germline in two y– F1
♂ recovered from an F0
♀ cross that also yielded y– female siblings. These y– F1
♂ were considered candidates for carrying the y-MCR
construct and were crossed to y+ females. All but one of their F2
female progeny had a full-bodied y– phenotype (Fig. 2, E and F
). Occasionally among yMCR
♀ we also recovered mosaics (~4%) with a few small y+ patches as well as a lone example of a 50% chimeric female (Fig. 2H
), and in two instances, we recovered y+ male progeny from a yMCR
♀ mother (Fig. 2E
and table S1). These infrequent examples of imperfect y-MCR
transmission indicate that although HDR
is highly efficient at this locus in both somatic and germline lineages, the target occasionally evades conversion.
Polymerase chain reaction (PCR
) analysis of the y locus in individual y– F1
progeny confirmed the precise gRNA- and HDR
-directed genomic insertion of the y-MCR
construct in all flies giving rise to y– female F2
progeny (Fig. 2D
). Males carried only this single allele, as expected, whereas females in addition possessed a band corresponding to the size of the wild-type y locus (Fig. 2D
, lane 4), which varied in intensity between individuals, indicating that females were mosaic for MCR
conversion. The left and right y-MCR PCR
junction fragments were sequenced from y– F1
progeny from five independent F0
parents. All had the precise expected HDR
-driven insertion of the y-MCR
element into the chromosomal y locus. In addition, sequence analysis of a rare nonconverted y+ allele recovered in a male offspring from a yMCR
♀ (Fig. 2E
) revealed a single-nucleotide change at the gRNA cut site (resulting in a T→I substitution), which most likely resulted from nonhomologous end-joining repair, as well as an in-frame insertion-deletion (indel) in a y+♀ sibling of this male (fig. S1 and table S1). The high recovery rate of full-bodied y– F1
female progeny from single parents containing a yMCR
allele detectable by PCR
indicates that the conversion process is remarkably efficient in both somatic and germline lineages. Phenotypic evidence of mosaicism in a small percentage of MCR
-carrying females and the presence of ylocus–derived PCR
products of wild-type size in all tested y– F1
females suggest that females may all be mosaic to varying degrees. In summary, both genetic and molecular data reveal that the y-MCR
element efficiently drives allelic conversion in somatic and germline lineages.
MCR technology should be applicable to different model systems and a broad array of situations, such as enabling mutant F1 screens in pioneer organisms, accelerating genetic manipulations and genome engineering, providing a potent gene drive system for delivery of transgenes in disease vector or pest populations, and potentially serving as a disease-specific delivery system for gene therapy strategies. We provide an example in this study of an MCR element causing a viable insertional mutation within the coding region of a gene. It should also be possible, however, to efficiently generate viable deletions of coding or noncoding DNA by including two gRNAs in the MCR construct targeting separated sequences and appropriate flanking homology arms. Using the simple core elements tested in this study, MCR is applicable to generating homozygous viable mutations, creating regulatory mutations of essential genes, or targeting other nonessential sequences. The method may also be adaptable to targeting essential genes if an in-frame recoded gRNA-resistant copy of the gene providing sufficient activity to support survival is included.
Video. Dr. Jennifer Doudna discusses the ethical implications of using CRISPR-Cas9
In addition to these positive applications of MCR
technology, we are also keenly aware of the substantial risks associated with this highly invasive method. Failure to take stringent precautions could lead to the unintentional release of MCR
organisms into the environment. The supplementary material includes a stringent, institutionally approved barrier containment protocol that we developed and are currently adhering to for MCR
experiments. Since this study was submitted for publication, a preprint has been posted on the bioRxiv web server showing that a split Cas9-gRNA gene drive system efficiently biases inheritance in yeast (6
). The split system was used to avoid accidental escape of the gene drives. The use of a similar strategy in future MCR
organisms would reduce, but not eliminate, risks associated with accidental release. We therefore concur with others (7
) that a dialogue on this topic should become an immediate high-priority issue. Perhaps, by analogy to the famous Asilomar meeting of 1975 that assessed the risks of recombinant DNA
technology, a similar conference could be convened to consider biosafety measures and institutional policies appropriate for limiting the risk of engaging in MCR
research while affording workable opportunities for positive applications of this concept.
Video. Conversations in Science with Dan Rather & Jennifer Doudna: CRISPR. Dr. Jennifer Doudna discusses the discovery of CRISPR-Cas9, ethical issues, her scientific career, and women in science.
Materials and Methods
References and Notes
F. Zhang, Y. Wen, X. Guo , CRISPR/Cas9 for genome editing: Progress, implications and challenges. Hum. Mol. Genet. 23, R40–R46(2014).
P. D. Hsu, E. S. Lander, F. Zhang , Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).
F. Port, H. M. Chen, T. Lee, S. L. Bullock , Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 111, E2967–E2976 (2014).
S. J. Gratz, F. P. Ukken, C. D. Rubinstein, G. Thiede, L. K. Donohue, A. M. Cummings, K. M. O’Connor-Giles , Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics 196, 961–971 (2014).
R. Bassett, C. Tibbit, C. P. Ponting, J. L. Liu , Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep. 4, 220–228 (2013).
J. E. DiCarlo, A. Chavez, S. L. Dietz, K. M. Esvelt, G. M. Church , http://biorxiv.org/content/early/2015/01/16/013896 (2015).
K. A. Oye, K. Esvelt, E. Appleton, F. Catteruccia, G. Church, T. Kuiken, S. B. Lightfoot, J. McNamara, A. Smidler, J. P. Collins , Regulating gene drives. Science 345, 626–628 (2014).
K. M. Esvelt, A. L. Smidler, F. Catteruccia, G. M. Church , Concerning RNA-guided gene drives for the alteration of wild populations. eLife 10.7554/eLife.03401 (2014).
Acknowledgments: We thank M. Yanofsky, W. McGinnis, S. Wasserman, R. Kolodner, H. Bellen, and members of the Bier lab for helpful discussions and comments on the manuscript; M. Harrison, K. O’Connor-Giles, J. Wildonger, and S. Bullock for providing CRISPR/Cas9 reagents and information; and J. Vinetz and A. Lubar for granting us access to their BSL2 Insectary. Supported by NIH grants R01 GM067247 and R56 NS029870 and by a generous gift from S. Sandell and M. Marshall. E.B. and V.M.G. are authors on a patent applied for by the University of California, San Diego (provisional patent application number 62075534) that relates to the mutagenic chain reaction. MCR fly stocks and DNA constructs are available from E.B. under a material transfer agreement from UCSD. This protocol for use and containment of our MCR stocks in a BSL2 barrier insectary also used for containment of malaria-infected mosquitos was reviewed and approved by the UCSD Institutional Biosafety Committee (BUA R461).