166CRISPR: hot, hot, hot

CRISPR is the latest technique for genome engineering and is generating tons of excitement due to its versatility, high specificity, and ease of use.

CRISPR stands for “clustered regularly interspaced short palindromic repeats”, but this has little to with how the technology is currently being used. In nature, CRISPR is part of a bacterial “immune system” that allows bacteria to recognize and destroy the DNA of viruses that attack it. The mechanism by which this happens is fascinating, but not very relevant to all of the current excitement.

First use of CRISPR for genome engineering reported in early 2013 in two papers which today have 865 and 811 citations, respectively:

Multiplex genome engineering using CRISPR/Cas systems. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Science. 2013 Feb 15;339(6121):819-23.

RNA-guided human genome engineering via Cas9. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. Science. 2013 Feb 15;339(6121):823-6.

There is a lot of controversy concerning the patent for this technology:http://www.technologyreview.com/featuredstory/532796/who-owns-the-biggest-biotech-discovery-of-the-century/

167CRISPR is the “bacterial

immune system”

Fig. 2. Overview of the CRISPR/Cas mechanism of action. (A) Immunization process: After insertion of exogenous DNA from viruses or plasmids, a Cas complex recognizes foreign DNA and integrates a novel repeat-spacer unit at the leader end of the CRISPR locus. (B) Immunity process: The CRISPR repeat-spacer array is transcribed into a pre-crRNA that is processed into mature crRNAs, which are subsequently used as a guide by a Cas complex to interfere with the corresponding invading nucleic acid. Repeats are represented as diamonds, spacers as rectangles, and the CRISPR leader is labeled L.

CRISPR/Cas, the Immune System of Bacteria and Archaea. Philippe Horvath, Rodolphe Barrangou, Science 8 January 2010, Vol. 327 no. 5962 pp. 167-170

Genome engineering 168

The key to genome engineering is to first introduce a double strand break at a specific location in the genome

If an appropriate piece of donor DNA is provided, which is homologous to the regions flanking the break, the break will be repaired by homologous recombination

and the donor DNA can be inserted. If there is no donor DNA, the break will be repaired by non-homologous end joining, which typically results in mutation.

“If a homologous donor DNA is provided (solid box, left), repair can proceed by homologous recombination using the donor as template. The amount of donor sequence ultimately incorporated will typically decline with distance from the original break, as illustrated by the shading. Alternatively, the break can be repaired by nonhomologous end joining, leading to mutations at the cleavage site. These may be deletions, insertions, and base substitutions, usually quite localized, but sometimes extending away from the break. ”

D. Carroll. Genome Engineering With Zinc-Finger Nucleases. Genetics. 2011; 188(4): 773–782.

The three genome engineering strategies 169

• RNA-guided human genome engineering via Cas9. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. Science. 2013 Feb 15;339(6121):823-6.

• Multiplex genome engineering using CRISPR/Cas systems. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Science. 2013 Feb 15;339(6121):819-23.

• Chimeric restriction endonuclease. Kim, Y.-G., and S. Chandrasegaran, 1994. Proc. Natl. Acad. Sci. USA 91: 883–887.

• Hybrid restriction enzymes: zinc finger fusions to FokI cleavage domain. Kim, Y.-G., J. Cha, and S. Chandrasegaran, 1996. Proc. Natl. Acad. Sci. USA 93: 1156–1160.

• Targeting DNA double-strand breaks with TAL effector nucleases. Christian, M, Cermak, T, Doyle, EL, Schmidt, C, Zhang, F, Hummel, A et al. (2010). Genetics 186: 757–761.

• Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Zhang, F.*, Cong, L.*, Lodato, S., Kosuri, S., Church, G.M. & Arlotta, P. Nat Biotechnol 29, 149-153 (2011).

https://www.addgene.org/CRISPR/guide/

D. Carroll. Genome Engineering With Zinc-Finger Nucleases. Genetics. 2011; 188(4): 773–782.http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3176093/

The accelerating pace of genome engineering170

The new frontier of genome engineering with CRISPR-Cas9. Doudna JA, Charpentier E. Science 2014 Nov 28;346(6213):1258096

Crystal structure of Cas9 in complex with guide RNA and target DNA

171

Crystal structure of Cas9 in complex with guide RNA and target DNA. H. Nishimasu, F.A. Ran, P.D. Hsu, S. Konermann, S.I. Shehata, N. Dohmae, R. Ishitani, F. Zhang, O. Nureki. Cell, 156 (2014), pp. 935–949

“Bacteria with a Type II system employ a single CRISPR-associated protein, Cas9, to generate double-stranded breaks in viral DNA or invading plasmids during an adaptive immune response. Cas9 is a multi-domain and multi-functional protein ( Figure 4b). It contains HNH-nuclease and RuvC-like nuclease domains, which cleave the DNA complementary strand (target strand) and the non-complementary strand (non-target strand), respectively [ 29•• and 52]. By forming Watson–Crick base pairs with the 3′-repeat region of mature crRNA, the tracrRNA:crRNA hybrid recruits Cas9 to cleave the foreign DNA guided by spacer sequences within crRNA [ 29•• and 52]. The dual-tracrRNA:crRNA guide RNA, when engineered as a single-guide RNA (sgRNA) chimera, enabled rapid implementation of CRISPR-Cas9 as a powerful tool for genome engineering [ 29••].”

The structural biology of CRISPR-Cas systems. Fuguo Jiang, Jennifer A Doudna,, Current Opinion in Structural Biology, 2015, Volume 30, Pages 100-111.

Cas9 Targeting and the CRISPR Revolution, R. Barrangou, Science 16 May 2014: Vol. 344 no. 6185 pp. 707-708

Crystal structure of Cas9 in complex with guide RNA and target DNA

172

The new frontier of genome engineering with CRISPR-Cas9. Doudna JA, Charpentier E. Science 2014 Nov 28;346(6213):1258096

Cas9 normally requires two pieces of RNA, the tracrRNA which mostly binds to the protein, and the crRNA which is specific for the target sequence. For engineering applications, these two pieces are fused together to make what’s called the single guide RNA (sgRNA)

The PAM sequence is a short sequence of DNA that must be immediately adjacent to the section of DNA that is being recognized by the crRNA. This sequence is important because it is a way for the bacteria to prevent its own DNA from being targeted. Presumably, the bacteria would not have the particular PAM sequence present anywhere in its own genome.

Catalytically inactive Cas9 (dCas9) plus appropriate gRNA, is proving very useful as a

general DNA-recognition module

173

https://www.addgene.org/CRISPR/guide/

Yet other applications of catalytically dead Cas9 include:1. Purifying specific regions of chromatin (i.e. for ChIP)2. Labeling regions of DNA in live cells for imaging (like live-cell FISH)

Current and future CRISPR technologies 174

CRISPR has already been used in many model organisms

Many people are concerned that we are moving too quickly towards human applications, and irreversible changes to wild populations

The new frontier of genome engineering with CRISPR-Cas9. Doudna JA, Charpentier E. Science 2014 Nov 28;346(6213):1258096

CRISPR Gene Drive technology 175

The ‘Gene Drive’ (or mutagenic chain

reaction) technology has the potential to

rapidly spread genomic changes

through whole populations.

http://www.sciencemag.org/content/early/2015/03/18/science.aaa5945

This technology could be used to make mosquito’s

resistant to malaria

The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations. Valentino M. Gantz*, Ethan Bier*. Science. Published Online March 19 2015. DOI: 10.1126/science.aaa5945. http://www.sciencemag.org/content/early/2015/03/18/science.aaa5945

See also:http://elifesciences.org/content/early/2014/07/15/eLife.03401http://biorxiv.org/content/biorxiv/early/2015/01/16/013896.full.pdf