A Guest Post from Thomas (TJ) Cradick, Director of the Protein Engineering Facility, Georgia Institute of Technology; @NucleaseLab

Genome editing has remained a therapeutic goal since before specific, disease-causing mutations were discovered. Introducing mutations into cell lines and model organisms have also created very useful research reagents. The rate of both processes is greatly enhanced by creating nearby DNA breaks. These effects were first shown with meganucleases, which are very specific but have proved very difficult to convert to targeting novel sequences. The first readily engineered nuclease group was ZFNs (zinc-finger nucleases), followed by TALENs (transcription activator-like effector nucleases), and most recently CRISPRs (clustered regularly interspersed short palindromic repeats). The DNA breaks caused by these nucleases are repaired by the cellular DNA repair machinery and can lead to precise modification. Genome editing is no longer science fiction, though issues remain on delivery and specificity.

TALENs are easier to design than Zinc Finger Nucleases due to straightforward rules linking DNA binding repeats to a target sequence [1,2]. These rules don’t help pick the highest activity sites, though a new program, SAPTA, helps pick sites that can be targeted with high activity and specificity [3]. Several groups have developed high-throughput cloning methods to assemble the DNA binding repeats in TALENs, though new proteins must be assembled for each target [4,5].

For each target, CRISPR systems have the advantage of using identical proteins identified as a means for bacteria to fend off pathogens. These gene-editing systems are called clustered regularly interspaced short palindromic repeats (CRISPR) and pronounced “crisper”. Cas (CRISPR-associated) proteins clone a piece of the foreign DNA into the CRISPR genomic locus between the repeats. Many of these foreign DNAs are saved in the daughter cells. One bacterial protein called Cas9 and a guide strand RNA expressed from these saved DNA pieces allow targeting complementary sequences if the foreign DNA is encountered again. The key is that CRISPR works by cutting DNA complementary to the “guide strand” RNA. Directing cleavage to a new target site only requires cloning a pair of annealed oligos into the guide strand expression cassette [6,7]. This saves the very difficult step of designing and cloning new DNA binding proteins, as are required for ZFNs or TALENs.

In the beginning of 2013 papers began describing genome editing in mammalian cells [7,8]. A number of labs made their plasmids available on Addgene and several created websites and online forums to spread the word. A new company, Editas Medicine, founded by five world leaders in genome editing was founded to use CRISPR and TALENS as treatments for genetic diseases.

One of the big concerns with each type of nuclease is “off-target mutation” in different region of the genome. Several programs help verify and optimize specificity by listing putative off-target cleavage sites, including PROGNOS for ZFNs and TALENs [9]. Others have also found ZFN and TALEN off-target sites, primarily through experimentally guided off-target searches. Currently, there are limited data indicating that TALENs have improved specificity over ZFNs and lower cytotoxicity [10-12]. A number of publications have described the high level of off-target cleavage possible using CRISPR [13,14] and chromosomal deletions or re-arrangements [15]. Although ZFNs and TALENs have off-target cleavage as well, the high levels seen with current CRISPR methods has many groups scurrying to develop newer, safer methods. Use of pairs of Cas proteins that each cut only one strand holds promise, though has yet to be optimized for gene repair [16,17]. It is also likely that improved CRISPR systems will be developed that provide more specificity, though they may have decreased targeting efficiency. For many applications, the ease and speed of the current generation of CRISPR systems will provide a valuable research tool while the work on CRISPR 2.0 continues.

References

1. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, et al. (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326: 1509-1512.

2. Moscou MJ, Bogdanove AJ (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326: 1501.

3. Lin Y, Fine EJ, Zheng Z, Antico CJ, Voit RA, et al. (2014) SAPTA: a new design tool for improving TALE nuclease activity. Nucleic Acids Research: gkt1363.

4. Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, et al. (2012) FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol 30: 460-465.

5. Schmid-Burgk JL, Schmidt T, Kaiser V, Höning K, Hornung V (2013) A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes. Nat Biotechnol 31: 76-81.

6. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816-821.

7. Cong L, Ran FA, Cox D, Lin S, Barretto R, et al. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819-823.

8. Mali P, Yang L, Esvelt KM, Aach J, Guell M, et al. (2013) RNA-guided human genome engineering via Cas9. Science 339: 823-826.

9. Fine EJ, Cradick TJ, Zhao CL, Lin Y, Bao G (2013) An online bioinformatics tool predicts zinc finger and TALE nuclease off-target cleavage. Nucleic acids research: gkt1326.

10. Tesson L, Usal C, Ménoret S, Leung E, Niles BJ, et al. (2011) Knockout rats generated by embryo microinjection of TALENs. Nature Biotechnology 29: 695-696.

11. Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, et al. (2011) Genetic engineering of human pluripotent cells using TALE nucleases. Nature Biotechnology 29: 731-734.

12. Mussolino C, Morbitzer R, Lutge F, Dannemann N, Lahaye T, et al. (2011) A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res 39: 9283-9293.

13. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, et al. (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol.

14. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol.

15. Cradick TJ, Fine EJ, Antico CJ, Bao G (2013) CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Research 41: 9584-9592.

16. Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, et al. (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol.

17. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, et al. (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154: 1380-1389.