Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.

http://stacks.cdc.gov/view/cdc/29957/cdc_29957_DS1.pdf

CRISPR-Cas systems for editing, regulating and targeting genomes

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

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DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

1. Jinek, M. et al. Science 337, 816–821 (2012). 2. Cho, S.W., Kim, S., Kim, J.M. & Kim, J.S. Nat. Biotechnol. 31, 230–232 (2013). 3. Cong, L. et al. Science 339, 819–823 (2013). 4. Mali, P. et al. Science 339, 823–826 (2013). 5. Hwang, W.Y. et al. Nat. Biotechnol. 31, 227–229 (2013). 6. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L.A. Nat. Biotechnol. 31, 233–239 (2013). 7. Wang, H. et al. Cell 153, 910–918 (2013). 8. Geurts, A.M. et al. Science 325, 433 (2009). 9. Tong, C., Li, P., Wu, N.L., Yan, Y. & Ying, Q.L. Nature 467, 211–213 (2010). 10. Tesson, L. et al. Nat. Biotechnol. 29, 695–696 (2011). 11. Wu, H. & Zhang, Y. Genes Dev. 25, 2436–2452 (2011). 12. Gu, T.P. et al. Nature 477, 606–610 (2011). 13. Dawlaty, M.M. et al. Dev. Cell 24, 310–323 (2013). revision process of this work, an independent study reported the simultaneous generation of multiple mutations in mice7. Our work, together with the mice work, demonstrates that it should be feasible to produce genetargeted models in rodents and probably other mammalian species using the CRISPRCas systems.

Targeted genome modification of crop plants using a CRISPR-Cas system.

RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants, but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. Here, we deliver purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells. RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off-target mutations associated with plasmid transfection at off-target sites that differ by one or two nucleotides from on-target sites. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects. Furthermore, RNP delivery is less stressful to human embryonic stem cells, producing at least twofold more colonies than does plasmid transfection.

/pdf/highly-efficient-rna-guided-genome-editing-in-human-cells-1dwagla9ju.pdf

Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins

Sequence-specific nucleases have been applied to engineer targeted modifications in polyploid genomes, but simultaneous modification of multiple homoeoalleles has not been reported. Here we use transcription activator-like effector nuclease (TALEN) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (refs. 4,5) technologies in hexaploid bread wheat to introduce targeted mutations in the three homoeoalleles that encode MILDEW-RESISTANCE LOCUS (MLO) proteins. Genetic redundancy has prevented evaluation of whether mutation of all three MLO alleles in bread wheat might confer resistance to powdery mildew, a trait not found in natural populations. We show that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistance to powdery mildew. We further use CRISPR-Cas9 technology to generate transgenic wheat plants that carry mutations in the TaMLO-A1 allele. We also demonstrate the feasibility of engineering targeted DNA insertion in bread wheat through nonhomologous end joining of the double-strand breaks caused by TALENs. Our findings provide a methodological framework to improve polyploid crops.

Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew

Rapid and Efficient Gene Modification in Rice and Brachypodium Using TALENs

Bacteria and archaea have evolved an adaptive immune system, known as type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, which uses short RNA to direct the degradation of target sequences present in invading viral and plasmid DNAs. Recent advances in CRISPR/Cas system provide an improved method for genome editing, showing robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci. It is the latest technology to modify genome DNA specifically and effectively following zinc finger nucleases (ZFNs) and TALE nucleases (TALENs). Compared with ZFNs and TALENs, CRISPR/Cas is much simpler and easier to engineer. This review summarizes recent progress, and discusses the prospects of CRISPR/Cas system, with an emphasis on its structure, principle, applications and potential challenges.

Jun Li

Papers

Targeted genome modification of crop plants using a CRISPR-Cas system.

Rapid and Efficient Gene Modification in Rice and Brachypodium Using TALENs

[CRISPR/Cas: a novel way of RNA-guided genome editing].

Site-directed modification method of rice genome