RNA-guided human genome engineering via Cas9

From Wiki FKKT

Jump to: navigation, search

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



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 the most cited and well respected scientists in the 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.[1]. 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[2]. 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[3].

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.


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[4] 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. PCR screen (see Figure 2C) confirmed that 21/24 randomly picked 293T clones were successfully targeted. Similar PCR screen confirmed 3/7 randomly picked29 PGP1‐iPS clones were also successfully targeted. Also, short 90-mer oligos could also effect robust targeting at the endogenous AAVS1 locus. In Supplementary Materials, Figure S10 we can se that NHEJ rate was 38%.

Bioinformatically generated gRNA-targetable sequences

This versatile RNA-guided genome engineerig system can be adapted to modify other genomic sites just by modifying the sequence of sgRNA expression vector used to match a compatible sequence in the locus of interest. To facilitate this process, authors bioinformatically generated ~190,000 specifically gRNA-targetable sequences targeting ~40.5% exons of genes in the human genome (shown in Supplementary Materials Table S1). These target sequences are incorporatedinto a 200bp format compatible with multiplex synthesis on DNA arrays (shown in Supplementary Materials, Figure S11 and tables S2 and S3). This work provides a ready genome-wide source of potential target sites in the human genome and a methodology for multiplex gRNA synthesis.

Other Cas9 prospects

Recent studies have shown possibilities of versatile Cas9 mediated use for genome editing and regulation. Great versatility and potential of the Cas9 as combining factor with ability to bring together DNA, RNA and proteins. Proteins can be targeted to any dsDNA sequence by simply fusing them to a nuclease-null Cas9 and expressing a suitable sgRNA. Note that some authors use “dead” Cas9 (dCas9) as term for Cas9 protein with lack of nuclease activity. Consequently, Cas9 can bring any fusion protein together with any fusion RNA at any dsDNA sequence by covalent attachment to dCas9 or to sgRNAs, or by noncovalent binding to covalently attached molecules. Knowing that effective concentration is important in regulation of biological processes, CRISPR/Cas9 system can be used as a single unifying factor, capable of mediating biologic interactions. Therefore, it has a great potential for use in investigating and engineering living systems. For example, transcription is dependent on the assembly of regulatory complexes and their interactions with chromatin. By targeting dCas9 to important binding sites for transcription factors, it should be possible to obstruct the binding of these factors and thereby exclude their role in transcription. Similarly, individual factors with unknown roles could be selectively recruited to almost any desired sequence by dCas9 fusions or sgRNA tethers with only slightly less precision. Together, these capabilities may allow a component-by-component approach to perturbing endogenous gene regulation[5].

Transcriptional activation

For engineering purposes, it is often useful to directly upregulate the transcription of endogenous genes to a desired level of activity. Experiments with zinc finger effectors and transcription activator–like (TAL) effectors demonstrated that multiple VP64 activator domains localized 5′ of the transcription start site yield synergistic effects. It was shown that Cas9-mediated localization functions similarly with dCas9–VP64. It is important to know that the rate of activation can vary among targeted genes. It requires synergy between multiple Cas9-sgRNA activators for robust transcription. Activation is probably dependent on local chromatin structure, unique interactions with endogenous transcriptional machinery and the Cas9 biochemistry. Elucidation of these effects as well as evaluation of additional Cas9 orthologs will be necessary for fine tuning of control over endogenous transcription. The capability to upregulate any endogenous gene or combination of genes in trans-acting manner has tremendous implications for ability to investigate and control cellular behavior. In particular, multiplexed sgRNA libraries targeting every known gene could help point out the factors responsible for important cellular processes, such as differentiation.

Transcriptional repression

Fusing of repressor domains to zinc finger effector or TAL effector proteins potently suppresses endogenous transcription. By using a similar architecture for dCas9–KRAB or related fusion proteins or sgRNA-based tethers, it should be possible to repress genes with equivalent efficacy and far greater ease of targeting. Indeed, a dCas9–KRAB fusion has been recently shown to induce modest repression using single guide RNAs. Localizing additional repressors and optimizing the structure of the fusion protein could greatly increase the potency of this approach. The ability to repress transcription will not only complement studies using transcriptional activation, but may also be useful for antiviral applications in eukaryotic cells. By preventing the transcription of invading viral genomes, Cas9 repressors could in principle “equip” a transgenic organism with immune to many DNA viruses, targeted with sufficient sgRNAs. This might be a great advantage for crops and domesticated animals.

Improving specificity

An increasingly recognized limitation in Cas9-mediated genome engineering applications is their specificity of targeting. The sgRNA-Cas9 complexes are in general tolerant of 1–3 mismatches in their target and occasionally more. It depends on the function of the Cas9 ortholog, the sgRNA architecture, the targeted sequence, the PAM, and also the relative dose and duration of these reagents. Although imperfect Cas9 specificity is a major reason for concern, there are several methods of potentially improving this drawback. Improvements include requiring multiple sgRNA-Cas9 complexes for activity, reducing affinity while increasing cooperativity, establishing competition between inactive and active forms, discovering improved natural orthologs, engineering improved variants and choosing targeting sgRNAs wisely.

Engineering Cas9-targeted recombinases

Despite the effectiveness of nuclease-based methods in editing genomes, safe in vivo gene correction in humans remains difficult. Most notably, the introduction of a double-strand break or even a nick at the wrong off-target site can lead to unexpected mutations or rearrangements that may have consequences in carcinogenesis. Site-specific recombinase, and potentially transposase enzymes present fewer problems by tightly controlling generation of DSBs to coordinate donor-target coupling. By fusing the catalytic domain of a small serine recombinase to Cas9, analogous to previous zinc finger and TAL fusions, it may be possible to create an RNA-guided recombinase enzyme. Because the activity of such retargeted fusion recombinases is generally low, extensive directed evolution may be necessary to produce a useful RNA-guided recombinase.

Discovering or evolving improved Cas9 proteins

It is possible that certain Cas9 orthologs might prove more specific than the Cas9 from S. pyogenes. It is unlikely that Cas9 proteins with longer PAM requirements will exhibit greater overall specificity, as the selective pressure for accurate recognition of the combined spacer and PAM remains constant. However, Cas9 proteins from species with larger genomes may be somewhat more specific, and those that have undergone frequent horizontal gene transfer along with their CRISPR loci and consequently been selected for avoidance of multiple host genomes are likely the most specific of all. The best Cas9 proteins identified in nature might be improved by rational design (usually by mutagenesis studies), directed evolution or ideally a combination of the two. One attractive strategy for improving specificity is to reduce the basal Cas9 affinity for DNA, which could be dimminished at target sites by employing two cooperatively binding sgRNAs with complementary 3′ overhangs that target adjacent protospacers. Alternatively, the PAM might be changed to expand the range of targetable sites or enlarged to increase specificity, although such alteration may not be accessible by rational design alone. PAM alteration and more complex modifications might be accessible using directed evolution, including increasing the overall specificity of each Cas9 monomer. Such experiments must be designed to select for activity at a perfectly matched protospacer. Activity at mismatched sites, preferably those identified as problematic by specificity measurement assays is also important. Ideally, the process would result in selection against many mismatched protospacers at any one time, and the process would be repeated over many rounds of selection. Methods of directed evolution would be convenient for this challenge[5]..


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[4] 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 of Streptococcus pyogenes Cas9 was published [6]. This achievement offers the possibility of rational engineering of 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 already desinging CRISPR reagents for the research community. A few already commercially available products are CRISPR online design tools, CRISPR paired nickases for high specificity genome editing and Cas9 mRNA and expression plasmids.[7].  


  1. 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
  2. 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
  3. 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
  4. 4.0 4.1 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.
  5. 5.0 5.1 Mali, P., Esvelt, K. M. & Church, G. M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–63 (2013)
  6. Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–49 (2014)
  7. Baker, M., 2014. Gene editing at CRISPR speed. Nat. Biotechnol. 32, 309–12. doi:10.1038/nbt.2863

SB students resources

Personal tools