RNA-guided human genome engineering via Cas9: Difference between revisions

From Wiki FKKT
Jump to navigationJump to search
No edit summary
No edit summary
Line 45: Line 45:
HR was observed only when introduction all three components of CRISPR/Cas9 system  were present (repair donor, Cas9 protein, and gRNA). This result confirms that all components are required for genome editing.
HR was observed only when introduction all three components of CRISPR/Cas9 system  were present (repair donor, Cas9 protein, and gRNA). This result confirms that all components are required for genome editing.
When mutating the target genomic site, sgRNA had no effect at HR in that locus, demonstrating that CRISPR/Cas9 mediated genome editing is sequence specific.
When mutating the target genomic site, sgRNA had no effect at HR in that locus, demonstrating that CRISPR/Cas9 mediated genome editing is sequence specific.
293T cells transfection with various combinations of constructs (humanized Cas9+T1 sgRNA and humanized Cas9+T2 sgRNA). NHEJ rates measurement (4 days after nucleofection) was performed by deep sequencing, detecting genomic deletions and insertions at DSBs. 293T targeting by both sgRNAs is efficient (10-24%) and sequence  specific. These results show that using T2 sgRNA yelds higher genome targeting rates. These result might be the consequence of local target DNA structure, due to better T2 sgRNA affinity of first 12 bp after PAM sequence.
293T cells transfection with various combinations of constructs (humanized Cas9+T1 sgRNA and humanized Cas9+T2 sgRNA). NHEJ rates measurement (4 days after nucleofection) was performed by deep sequencing, detecting genomic deletions and insertions at DSBs. 293T targeting by both sgRNAs is efficient (10-24%) and sequence  specific. These results show that using T2 sgRNA yelds higher genome targeting rates. These result might be the consequence of local target DNA structure, due to better T2 sgRNA affinity of first 12 bp after PAM sequence. Jinek ''et al''<ref name="ref3">Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., Doudna, J., 2013. RNA-programmed genome editing in human cells. Elife 2, e00471. doi:10.7554/eLife.00471.</ref> suggested that target sites must perfectly match the PAM sequence NGG and the following 8-12 base at the 3′ end of the gRNA.





Revision as of 14:45, 11 January 2015

Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E., Church, G.M. RNA-guided human genome engineering via Cas9. Science 339, 823–6. 2013

Luka Smole

Results figure 1
Results figure 2
Supplementary Materials

Introduction

In this seminar I will write about CRISPR/Cas system, in this case constructed for human genome engineering. In this research authors engineered bacterial type II CRISPR system to target endogenous AAVS1 locus in human cells with two different sgRNAs to promote homologous recombination. The article I will focus on was published in Science by Prashant Mali from George M. Church’s research group from Harvard Medical School in Boston, Department of Genetics in 2013. George M. Church is one of most cited and well respected scientists in field of synthetic biology. Since 2013, his research group published many papers on CRISPR/Cas system in highly respected journals such as Nature biotechnology, Nucleic acids research and Science.


  • Note:

While writing this seminar, I put a lot of effort in summarizing this research in most comprehensible manner. To read this seminar in less confusing way, I suggest opening the links to figures at the very beginning of reading to better understand design of experiments and results (all important results are summarized in two figures).

CRISPR/Cas9 system

Making specific changes in DNA, such as changing, inserting or deleting sequences that code for proteins allows us to engineer cells, tissues and organisms for therapeutic and practical applications. Until now, such genome engineering has required the design and production of proteins with the ability to recognize a specific DNA sequence, such as zinc fingers and TALE (transcription activator like effector) proteins. Such techniques are relatively time consuming and expensive (especially on large scale, such as engineering for therapeutic applications). Thus, research of alternative strategies for triggering site-specific DNA cleavage in eukaryotic cells was in great interest. The bacterial protein, Cas9, had the potential to enable a simpler approach to genome engineering. It is a DNA-cleaving enzyme that can be programmed with single guiding RNA molecules (sgRNA) to recognize specific DNA sequences. This way, there is no need to engineer a new protein for each new DNA target sequence.<ref name="ref1">Sternberg, S.H., Redding, S., Jinek, M., Greene, E.C., Doudna, J. a, 2014. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–7. doi:10.1038/nature13011</ref>. This single RNA–single protein CRISPR system is derived from a natural adaptive immune system in bacteria and archaea. Prokaryotes have evolved diverse RNA-mediated systems that use short CRISPR RNAs (crRNAs) and Cas (CRISPR-associated) proteins to detect and defend against foreign DNA, such as phage DNA. Bacteria harbouring CRISPR/Cas loci respond to viral and plasmid challenge by integrating short fragments of the foreign nucleic acid (protospacers) into the host chromosome at one end of the CRISPR locus. The transcript of CRISPR loci is short CRISPR RNAs (crRNAs) that direct Cas protein-mediated cleavage of complementary target sequences within invading foreign (viral or plasmid) DNA. In type II CRISPR/Cas systems, Cas9 functions as a RNA-guided endonuclease that uses a dual-guide RNA. Guide RNA consists of crRNA (which interacts with Cas9 protein by “handle”) and trans-activating crRNA (tracrRNA) for target recognition and cleavage by a mechanism involving two nuclease active sites that together generate doublestranded DNA breaks (DSB). For schematic representation of CRISPR/Cas system, see this figure: [1]. As stated above, zinc fingers and TALEs are powerful tools in synthetic biology, but there are some drawbacks, because it remains time consuming and expensive to develop large-scale protein libraries for genome interrogation<ref name="ref5">Gilbert, L. a, Larson, M.H., Morsut, L., Liu, Z., Brar, G. a, Torres, S.E., Stern-Ginossar, N., Brandman, O., Whitehead, E.H., Doudna, J. a, Lim, W. a, Weissman, J.S., Qi, L.S., 2013. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–51. doi:10.1016/j.cell.2013.06.044</ref>. Note that authors use different terms for guiding RNAs in CRISPR systems due to lack of established terminology on this relatively new field of synthetic biology. So far the popular terms are short-guide and single-guide RNA, but they mean the same RNA that “guides” Cas9 nuclease to target DNA sequence. Some authors refer crRNA/tracrRNA complex as single-guide RNA or sgRNA (because it works for both Cas9 binding and DNA target site recognition as single transcript).


Homologous recombination (HR), non-homologous end joining (NHEJ)

It is important to be familiar with mechanisms of homologous recombination (HR) and non-homologous end joining (NHEJ) for understanding the design and principle of this human genome engineering study. We must point out that HR is a process that uses a desired homologous repair “primer” of donor DNA as a template from which it copies the information, which was lost during DSB. NHEJ on the other hand simply joins ends without homology and often results in deletions and/or insertions. These mechanisms are well shown: here<ref name="ref4">Jeggo, P. a, Löbrich, M., 2007. DNA double-strand breaks: their cellular and clinical impact? Oncogene 26, 7717–9. doi:10.1038/sj.onc.1210868 </ref>.


Design of CRISPR/Cas9 system for RNA-guided human genome engineering

In this research 1 engineered bacterial type II CRISPR system to target endogenous AAVS1 locus in human cells with two different sgRNAs to promote homologous recombination. They synthesized a human codon-optimized version of the Cas9 protein bearing a C terminus SV40 nuclear localization signal and cloned it into a mammalian expression system. Here is a plasmid map of this construct: https://www.addgene.org/41815/. To direct Cas9 to specific sequences of interest, single guide RNAs (sgRNAs) was expressed from the human U6 polymerase III promoter. Schematic representation of construct designs is shown of figure 1 A: [2]. The first important constrain is that U6 transcription must initiate with G. The second constrain in all CRISPR/Cas systems is the requirement for the PAM (protospacer-adjacent motif) sequence -NGG following the ≈20 bp sgRNA target. Regarding the mentioned facts, CRISPR/Cas9 system can in principle target any genomic site of the form G(N)20GG. They developed a GFP reporter assay to test the functionality CRISPR/Cas9 system as genome engineering tool. To test the efficiency of system at stimulating HR, two sgRNAs (T1 and T2) that target the intervening AAVS1 fragment were constructed (Figure 1 B [3]). A genomically integrated GFP coding sequence is disrupted by the insertion of a stop codon and a 68bp genomic fragment from the AAVS1 locus. Restoration of the GFP sequence by homologous recombination with an appropriate donor sequence results in GFP+ cells enabling quantification by FACS (flow activated cell sorting). HR stimulation rate was then compared to TAL effector nuclease heterodimer (TALEN) targeting the same region.


Results

Targeting GFP reporter system

Figure 1

Successful HR events were observed when targeting previously described GFP reporter system. Gene correction rates were 3% when T1 sgRNA and 8% when T2 sgRNAs was used in CRISPR/Cas9 system. This system has proved to be more rapid than TALENs with the first GFP+ cells appearing ~20 hours post transfection while ~40 hours for the TALENs. HR was observed only when introduction all three components of CRISPR/Cas9 system were present (repair donor, Cas9 protein, and gRNA). This result confirms that all components are required for genome editing. When mutating the target genomic site, sgRNA had no effect at HR in that locus, demonstrating that CRISPR/Cas9 mediated genome editing is sequence specific. 293T cells transfection with various combinations of constructs (humanized Cas9+T1 sgRNA and humanized Cas9+T2 sgRNA). NHEJ rates measurement (4 days after nucleofection) was performed by deep sequencing, detecting genomic deletions and insertions at DSBs. 293T targeting by both sgRNAs is efficient (10-24%) and sequence specific. These results show that using T2 sgRNA yelds higher genome targeting rates. These result might be the consequence of local target DNA structure, due to better T2 sgRNA affinity of first 12 bp after PAM sequence. Jinek et al<ref name="ref3">Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., Doudna, J., 2013. RNA-programmed genome editing in human cells. Elife 2, e00471. doi:10.7554/eLife.00471.</ref> suggested that target sites must perfectly match the PAM sequence NGG and the following 8-12 base at the 3′ end of the gRNA.


Targeting in native endogenous AAVS1 locus

Figure 2

After successful targeting of integrated reporter, the next goal was to modify a native locus. SgRNAs to target the AAVS1 locus were used (described above in paragraph Design of CRISPR/Cas9 system for RNA-guided human genome engineering). AAVS1 locus is located in the PPP1R12C gene on chromosome 19, which is ubiquitously expressed across most tissues. Genome modification tests were performed in 293T, K562, and PGP1 human iPS cells. Results were analyzed by next-generation sequencing of the targeted locus. As in previous experiments of targeting the GFP reporter assay, authors have observed high rates of NHEJ at the endogenous locus for all three cell types. The two gRNAs, T1 and T2, achieved NHEJ rates of 10 and 25% in 293Ts, 13 and 38% in K562s, and 2 and 4% in PGP1-iPS cells, respectively (Fig. 2B). As observed, NHEJ rates vary in cell types, most probably due to different complex endogenous processes. As seen on figures, total count and location of deletions caused by NHEJ for T1 and T2 were centered around the target site positions. These results clearly show, once more, the sequence specificity CRISPR/Cas9 system. Simultaneous introduction of both T1 and T2 gRNAs resulted in high efficiency deletion of the targeted 19bp fragment, demonstrating that multiplexed editing of genomic loci is feasible using this approach.


Modifying the native AAVS1 locus

Using CRISPR/Cas9 system to induce HR for integration of donor construct or an oligo donor into the endogenous loci in human cells has a great potential for future therapeutic use. Authors confirmed HR-mediated integration in the native AAVS1 locus using both approaches by PCR and Sanger sequencing.

Conclusion

These results show that RNA-programmed genome editing is a straightforward strategy for introducing site-specific genetic changes in human cells. Due to ease of design and effectiveness, in 2013 CRISPR/Cas system took over most the researcher’s attention in field of tools for genome regulation and modification. It seems that it is likely to become competitive with existing approaches based on zinc finger nucleases and transcription activator-like effector nucleases, and could lead to a new generation of experiments in the field of genome engineering for such complex genomes as human<ref name="ref3">Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., Doudna, J., 2013. RNA-programmed genome editing in human cells. Elife 2, e00471. doi:10.7554/eLife.00471.</ref> Research into the CRISPR-Cas gene editing system continues at great speed. The ease, low cost and speed of designing an RNA guided endonuclease against a DNA target of interest has caught the imagination of worldwide researchers. In beginning of 2014, the crystal structure Streptococcus pyogenes Cas9 was published 6. This achievement offers the possibility of rational engineering of the this RNA-protein complex based on structural information for the first time. The interest is not in academic sphere; several startups have been created around the technology. Also, reagent companies are rushing to create CRISPR reagents for the research community<ref name="ref2">Baker, M., 2014. Gene editing at CRISPR speed. Nat. Biotechnol. 32, 309–12. doi:10.1038/nbt.2863</ref>.  

References

<references />

SB students resources