Move over, Cas9: Cpf1 may be your new #CRISPR competition.

This past weekend at the Pilgrimage music festival in Franklin, TN I had the pleasure of seeing Weezer rock out, then I walked a few hundred yards to another stage to watch Wilco do the same.  These bands each had their own stage, but in the CRISPR world, Cas9 is a superstar that now has to share the stage with a newcomer:  Cpf1.  (Yeah, I know that's a goofy setup but it really was a good festival and it was on my mind.  Now on to the science.)

Last Friday Feng Zhang’s group published a paper in Cell that immediately grabbed a lot of attention, and rightly so.  They reveal that the Cpf1 class of CRISPR effector proteins may be an attractive alternative to Cas9.   Although Cpf1 has many similarities to Cas9, it has some significant differences that are very interesting – and could lead to improved efficiencies for some types of gene editing.  

Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System.  Zetsche et al., 2015, Cell 163, 1–13October 22, 2015  (Avail. online Sept. 25 2015).

Here is my summary…First, they reviewed some background on CRISPR systems in bacteria to cover the basis of the study.   There are two major classes of CRISPR systems based mainly on the proteins involved; the cleavage effectors of class 1 are complexes of multiple proteins, while the class 2 effectors are single proteins like Cas9.  Within class 2 there are two subtypes of systems: those with Cas9, and another that has Cpf1.   Cpf1 means “CRISPR from Prevotella and Francisella 1”.  Some bacterial species carry both Cas9-CRISPR and Cpf1-CRISPR loci in their genomes.   Like Cas9, Cpf1 has RuvC-like DNA cleavage domains but it lacks some of the other domain and neighbor-gene features of Cas9, so it’s clearly distinct in its evolution.    It’s a largish protein of ~1300 amino acids, similar in size to Cas9.

They picked the Cpf1-CRISPR gene system of Francisella novicida strain U112 to study first since there were clear homologies of Cpf1-CRISPR spacer sequences to various prophage in this species – further suggesting that Cpf1 is important in bacterial immunity and so it’s well adapted to slice and dice target DNAs.   (Sidebar: what’s F. novicida? A pretty rare human pathogen, originally isolated from the Great Salt Lake in Utah.  It’s related to the better known bug F. tularensis which is one of the most infectious pathogens known.)     

By transferring the F. novicida Cpf1-CRISPR gene locus into E. coli they quickly established that it prefers a  “TTN” PAM motif that is located 5’ to its protospacer target – not 3’, as per Cas9.  So right away it’s distinct in having a PAM that isn’t G-rich and is on the opposite side of the protospacer. 

Like Cas9, Cpf1 binds a crRNA that carries the protospacer sequence for base-pairing the target.  But for me the biggest surprise in the paper is that unlike Cas9, Cpf1 does not require a separate tracrRNA – in fact, there’s no sign of a tracrRNA gene at the Cpf1-CRISPR locus.   Thus, Cpf1 merely needs a cRNA that is about 43 bases long –of which 24 nt is protospacer and 19 nt is the constitutive direct repeat sequence.   This is very different than Cas9 – even by fusing the crRNA and tracrRNA, the single RNA that Cas9 needs is still ~100 nt long.

Furthermore, the Cpf1 crRNA does not have the long stemloop structure that is typical of RNAseIII-mediated processing to cut it out of its primary transcript.   It has a much shorter stemloop that is required for Cpf1 activity, however.   But surprisingly, Cpf1 itself is apparently directly responsible for cleaving the 43-base cRNAs apart from the primary transcript in the first place! This isn’t conclusively proven yet, but is pretty likely based on their experiments.   

Next, two more surprises comes from the cleavage sites on the target DNA.  The cut sites are staggered by about 5 bases.  This should create “sticky overhangs” that might be exploitable to enable gene editing via NHEJ-mediated-ligation of DNA fragments with matching ends.   And, the cut sites are in the 3’ end of the protospacer, distal to the 5’ end where the PAM is.    The cut positions usually follow the 18^th base on the protospacer strand and the 23^rd base on the complementary strand (the one that pairs to the crRNA).

They tested if they could inactivate the DNA cleavage domains via homologous mutations in codons known to do this in Cas9.  However, the resulting Cpf1 mutants don’t have “nickase” activity – they can’t cut either strand.   So it’s not clear that Cpf1 nickases can be made and in fact the authors suggest that the cleavage might require some sort of dimerization.  I can’t visualize how that would work yet but I’m sure it will be figured out in the near future…

Base substitution experiments then showed that, as per Cas9 CRISPRs, there is a “seed” region close to the PAM in which single base substitutions completely prevent cleavage activity.   Therefore, unlike the Cas9 CRISPR target the cleavage sites and the seed region do not overlap.  This immediately suggests a potential improvement over Cas9 in mammalian HDR-mediated repair efficiency.    This is because any initial cleavage events that might lead to “simple” NHEJ indels might still be substrates for cleavage  - and thus allowing additional chances for HDR-mediated edting to occur.  With Cas9, an indel mutation will almost always disrupt the target seeds and then it’s game over for HDR.     

Finally, they did the important work to screen various Cpf1 proteins from different bacterial species to see if any would actually work in mammalian cells.  This is because that despite codon optimization and attachment of nuclear localization signals, most of these bacterial proteins just don’t work right when you put them inside human or mouse cells.  Therefore they tested 16 different Cpf1 proteins.  Of these, for seven proteins they could identify PAM signatures using their E. coli assay.  They all had similar T-rich PAMs. 

Of these seven proteins, only two worked well in human HEK293 cells – AbCpf1, from an Acidaminococcus, and LbCpf1, which is from a Lachnospiraceae; interestingly these are apparently both anaerobic bacteria sometimes found in mammalian intestines.  Anyway these can both generate indels at specific targets in human cells at a rate similar to Cas9 – typically, 10-20% Surveyor assay numbers were observed, when they tested HEK cells following simple transfections.

Bottom line: Cpf1 may be the real deal as a serious competitor for Cas9.  Is Cas9 suddenly obsolete?  Hardly.  First, we don't yet know if Cpf1 is as specific as Cas9 – though there is every reason to think it may be.   So the off-target effects need to be carefully measured. Second, we don’t know how widespread the targeting efficiencies will be across sites (although the initial tests seem very promising).  Third, although Cpf1 may be better than Cas9 for mediating insertions of DNA, it’s not yet been shown if that is true.   However it may have some nice advantages over Cas9, not the least of which is that its guide RNA is only 43 bases long.  It will thus be feasible to purchase directly synthesized guide RNAs for Cpf1, perhaps with chemical modifications to enhance stability.

Probably, Cpf1 and Cas9 will both be in the spotlight for a long while to come.  Look for more Cpf1 papers to start coming out very soon and for Cpf1 plasmids to appear in Addgene.  Happy CRISPRing, everyone.

The reported off-target effects in the recent Liang et al human embryo #CRISPR paper are partly incorrect.

As widely reported last week, a group in China has published results of CRISPR editing experiments in human triponuclear embryos (Liang et al, Protein & Cell 2015).   The news blurb in Nature is worth a read to get the context of the paper, which follows on the heels of a previous statement published in Science by leaders in the CRISPR field and others, in which they discourage CRISPR experiments in human embryos at this time pending further discussion of the implications of such research.    

In this post I won’t get into the ethical implications of the paper (which is more than I can deal with in one post anyway!).   Here I’ll discuss the technical results.   The Liang et al paper does not actually present much data that is very surprising - it is not unexpected that CRISPR can induce targeted mutations in humans, since it works in basically every species in which it’s been tried.    Their target gene was HBB (beta-globin) and they attempted HDR using the familiar approach of coinjecting Cas9 mRNA, guide RNA, and donor oligo ssDNA.

Here’s their 4 main points, paraphrased from the abstract:   

1. Efficiency of HDR was low.
2. Edited embryos were mosaic.   
3. Off-target mutations were evident.
4. A separate, highly homologous gene (HBD) could serve as donor template for repair, thus introducing sequences inadvertently from the other gene into the target gene.

Of these, points #1 and 2 were not surprising to those who have injected CRISPR reagents into mouse embryos, and not all that different.   The reported HDR efficiency was 14%, which is in line with at least some published mouse experiments (e.g. Singh et al 2014).   I will state that in our mouse core we are apparently seeing HDR results around 10-20% efficiency across several experiments.    Mosaicism has also been previously reported in CRISPR mice (Yen et al 2014).  Point #4 was kind of novel, but in retrospect not completely weird, since the HBD gene (delta-globin) is over 90% identical to HBB.

Ok, so regarding point #3 - off-target mutations...I was very interested in this because the authors reported four distinct off-target (OT) mutations in human embryos associated with the single CRISPR guide RNA they used, and this has already been described in the media as being substantially higher than OT rates in animal embryos.  Meaning, mouse embryos.      

One of these OTs was particularly surprising, as it looked like a very poor match to the target protospacer indeed - although the 3’-most 11 bases (the “seed” region”) matched the target, the 5’ bases only matched 1 out of 9 bases, for a total of 8 mismatches.    Frankly, this really scared me, because if true it means that the current methods used to predict OTs are not nearly broad enough.   But this level of OT mismatch was much greater than any OT I had seen before.

Bottom line: After looking at their data, I now firmly believe that only one of the four OT mutations were actually new mutations caused by CRISPR.  The other three were simply polymorphisms, already present in the  germline, that the authors mistakenly classified as OTs.

Here is how they did their OT analysis and my interpretation of the data.   Tripronuclear human embryos were obtained from a fertility clinic; you can identify these microscopically at the 1-cell zygote stage.  Fertilized by 2 sperm by accident, they are effectively triploid, and are absolutely unable to survive to term as normal pregnancies - but they can survive well enough during short-term CRISPR experiments, in which the embryos are only kept alive for a few days in vitro.   Briefly, 86 tripronuclear embryos were injected; 71 survived the injection; 56 of these were GFP-positive (used to as a reporter to show expression of injected reagents) and used for DNA analyses of on- and/or off-target effects.   28 of these embryos had on-target indel mutations and/or the desired HDR edit and were used for OT analysis.   

Note that they had originally chosen this particular CRISPR target from 3 potential targets they looked at in their gene;  one of these didn’t cut well and was not used further . For each of the other two, 7 sites were identified as the “top” potential OTs by using the MIT tool.  Of the two targets, one was found to have no OT mutations at the 7 potential OT sites when it was tested in 293T cells.  So they decided to work with this CRISPR target.  

2 of the 7 OT’s were found to have mutations in the injected embryos.  These were named G1-OT4 and G1-OT5 and are the first two of the four total OT mutations they claimed to identify (Figure 3A).  T7 mismatch assays were used for this analysis.   293T cell transfections with the guide RNA had already shown a lack of mutations across the 7 OT sites, that is, they were negative by T7 assays.  I’ll come back to these later.

They then did whole-exome sequencing on six of the embryos to identify potentially even more mutated OTs.  From this data, they first called indels and SNVs (single nucleotide variants) and then searched for protospacer similarity “allowing for ≤6 mismatches or perfect match of the last 10 nt 3′ of the gRNA” anywhere within 100 bp of the indels.  (Not sure if they did anything more with the SNVs.)   This identified two apparently new OTs, in the 3’ UTRs of  the C1QC and TTR genes, each found in one embryo (Figure 3B).  These were confirmed by T7 assays.

So - what does the T7 mismatch assay really indicate?  It reveals heterozygosity within the PCR product.   Of course, new mutations can cause this.  But so can plain old polymorphisms.   This is a drawback of using mismatch assays when applied to polymorphic samples.     

The next question is, simply, are there common human polymorphisms in the PCR products used in the OT analysis?  It’s easy to check this using the UCSC genome browser and the 1000 genomes site.   

For OT #1, a.k.a. “G1-OT4”, (Fig. 1C and 3A; PCR, hg19, chr11:132761837-132762356; intron of OPCML) there are no known common polymorphisms within the PCR that are close to the OT.    The closest SNP, rs79549129, has a minor allele frequency (MAF) of 1.2% but zero in asian populations.   There are no other annotated variants near the OT with a significant MAF.  The closest “common” SNP is rs2659601 but it’s about 50 bp from one end of the PCR product.  I don’t think that could produce the band sizes seen in the Fig. S3 T7 assays. From what I can tell from their Fig. S3 & S4, their T7 assays are compatible with new mutations that have been induced by cleavage close to the CRISPR OT site.   Thus, these look like “real” CRISPR OT effects at this site.    6/20 of on-target embryos, or 30%, had mutations at this OT.  So this looks like a real OT effect that replicates across embryos, but not in 293T cells.   

But then, polymorphisms become apparent in the other OTs...

For OT #2, a. k. a. “G1-OT5”, (Fig. 1C and 3A; PCR, hg19 chr22:31000551-31001000; intron of TULP4), there are two common SNPs on either side of the OT:  rs616358 (G/C) and rs628203 (T/C).   Haplotype derivations in Southern Han Chinese suggest haplotype population frequencies of ~56% GT, 35% GC, 9% CT, and zero % CC.  So we would expect to see plenty of heterozygosity in this PCR - easily observable by T7 assays, in the range of 50% or so being positive in a population based sample from this geographic location  - no CRISPR required.  This OT was a false positive.  

UCSC screen grab showing SNPs close to the OT (black bar in middle)

This leaves the two additional OTs they discovered by whole-exome sequencing.  Remember their workflow: they called indels in their data sets, then looked for nearby partial matches to the CRISPR target.   However, they apparently did not filter out known polymorphisms first.

OT #3 was in the 3’ UTR of the TTR gene.   Inspection of the OT sequence location (given in Fig. S6) on the UCSC browser clearly shows that rs143948820 is a known 9-base indel contained completely inside the OT.       Turns out that it’s uncommon outside of Asia but it has a MAF of ~2% in Southern Han Chinese.  With a heterozygosity of ~4% in normal diploids, it’s totally possible that 1 out of 6 triploid embryos would carry this variant.  This OT was very likely a false positive. 

UCSC screen grab; OT is black bar, indel variant is long red bar.

Finally, OT #4 was in the 3’ UTR of the C1QC gene.   And similar to the case above, this OT overlaps with a known 17-base indel, rs142916975, that has a MAF of 38% in Southern Han Chinese.   Heterozygosity should be close to 50%.   In fact, I’m surprised they got a false positive in only 1 of their 6 samples.   This is almost certainly a false positive.

UCSC screen grab; OT in black, indel variant in blue.

In summary, only 1 of the OTs holds up to scrutiny.    Importantly, neither OT found by exome sequencing holds up.  This flips their conclusion on its head: “Our whole-exome sequencing result only covered a fraction of the genome and likely underestimated the off- target effects in human 3PN zygotes.”.   While it’s certainly possible that some more OTs could be found by whole genome sequencing, the exome data was essentially totally negative.   Note that they chose a CRISPR to work with because it had a low apparent OT rate in 293T cells.   In retrospect, it was just by luck that G1-OT5 has a negative T7 assay in 293T cells.  It could have been heterozygous, but it's apparently not.

To their credit, the authors rightly restate that it’s going to be critical moving forward to carefully analyze off-target effects in any human applications of CRISPR.  This is widely agreed upon (see below).  However this paper made some technical mistakes in this regard.  While underestimating off-target effects could certainly have serious negative consequences for future CRISPR-based clinical treatments for genetic disease - and nobody wants that - overestimating them could generate an excess of hesitation to research the feasibility of such treatments within the broader scientific community.  

As stated by Baltimore et al in their Science commentary:

“It is critical to implement appropriate and standardized benchmarking methods to determine the frequency of off-target effects and to assess the physiology of cells and tissues that have undergone genome editing.”

I don’t think I’m blowing smoke here that we all need to get this right, as the media quickly reported on the Liang et al conclusions:  

From Wired:  “But—and this is a big but—using the technique without proper guidance could result in unforeseen consequences. The Chinese researchers, for example, found mutations in many of the embryos in genes other than the ones they’d targeted with CRISPR/Cas9.”

From Time, quoting Carl Zimmer from National Geographic:   “The experiment “came out poorly,” Zimmer says; in some cases, DNA was placed in the wrong spot and “off-target” mutations were discovered in the DNA.”

From the Washington Post:   “And in some of the embryos, the gene editing caused unintended mutations in other genes.”

From USA today (emphasis is mine): “The team also found that the complex used in the procedure was also acting on other parts of the genome, leading to other bits of it mutating. That happened much more than in previous experiments on adult human cells and animal embryos — and could happen yet more if the whole genome were used, as it would be if the embryo were to be implanted.”

            (OK, note from this last article the specific comparison to the very observations that I have blogged about in more detail than most people probably ever wanted to hear about...My point is that, due to the technical problems in the Liang paper, I don’t think we can yet say the off-target effects were “much more than in previous experiments on adult human cells and animal embryos”. )

One final note - my analysis of this paper should not be interpreted to mean that I fully endorse CRISPR experimentation or applications in human embryos.   I also applaud the authors' cautionary tone that the incomplete efficiency of CRISPR editing in humans is a problem that any therapeutic applications need to address.

Whew, this was the longest post yet.   

COSMID, a tool to find #CRISPR off-targets including indels (usually not found w/other tools).

From Georgia Tech comes a new web tool, COSMID, for finding CRISPR off-targets (OTs).  But, this one definitely adds something new to the already busy realm of OT-finding tools: the ability to find OTs with small insertions or deletions relative to the target, not just base-pair mismatches.   Like "mismatched" OTs, off-target cleavage can occur at "indel-OTs" as well; see Lin et al, Nucl. Acids Res. (17 June 2014) 42(11): 7473-7485.    

COSMID is described in a new publication by T.J. Cradick, et al.  COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites, Molecular Therapy—Nucleic Acids (2014) 3, e214.    I've only used it briefly so far but it has a nice interface that allows the user to search for OTs with a user-specified max number of mismatches in combination with single-base insertions and/or deletions.  It also can perform PCR primer design for the OTs with the primers optimized for Surveyor-style mutation screening.    Thanks go to T.J. and colleagues for the nice addition to the CRISPR off-target screening toolkit.

Alas, this already raises a new caveat about my previous post reviewing off-targets in mice: I'm pretty sure that most or all of those citations did not look at indel OTs, only mismatch OTs.    !

Here's my review of published #CRISPR off-target mutation data from mouse embryo injections.

Here is a literature review of CRISPR off-target (OT) mutation analysis in mouse oocytes.    This review only concerns published experiments using “native” Cas9 that cuts both DNA strands, and not nickase-Cas9 experiments.  Although nickase-Cas9 is much less prone to OT mutation, editing is still generally less efficient than with native Cas9 .  Therefore it’s important to know whether the potential problem of off-target mutation rates with native Cas9 outweigh its utility.   All the data below is pertinent to mouse zygotes.  Other systems such as cell lines may have different OT rates.

Here’s a few pertinent questions to preface this review:  First, how should potential off-targets (OTs) be defined ahead of time?  It’s complicated by the fact that mismatches are less tolerated within the “seed” region of 8-12 bases proximal to the PAM site, and more tolerated in the more distal (5’) region of the protospacer.   So some groups define OTs as having perfect matches to the seed region, while other groups defined them as simply having fewer than a threshold maximum number of mismatches anywhere in the protospacer.   Alternatively, they can be scored for cleavage potential by algorithms such as the MIT CRISPR design tool.

Second, how is CRISPR performed? Some of these groups used RNA or DNA injections;  most used slightly varying injection concentrations.   

Third, how were the OTs screened?  Most of these did direct sequencing on PCR from founders or Surveyor-type assays.   Also, OTs often cut at lower efficiency than the on-target but the results depend on the assay sensitivity and the number of pups screened, which varies across studies.   So the data here is only a general comparison.

I’m not focusing on those differences here, since the overall picture is broadly similar - OT rates were generally low to nil.   

Let’s start with the pair of 2013 Cell papers from the Jaenisch lab.   

1.  Wang et al. (Cell 2013) was the first report of CRISPR-mediated mutagenesis in mouse zygotes.   They only considered OTs with perfect matches to the 12 bases adjacent to the PAM and also the PAM itself (NGG).  For 2 targets, they defined 7 total OTs. (A third gRNA they used had no OTs by this definition). In 7 mutants pups carrying mutations at 2 simultaneously-targeted gRNA targets, they found zero mutations at the 7 OTs.   

Bottom line:  7 OTs screened, 0 mutated.

2.  Yang et al (Cell 2013) screened OTs that were defined at having “up to 3 or 4” mismatches.  (In my experience most mouse CRISPR targets have several-to-many 3-base mismatches, and I’m guessing that most targets will have many 4-base mismatches in mammalian genomes.)  I believe they screened OTs for 5 targets across 4 different genes.   A total of 35 pups and ES cell clones were screened in separate experiments using different gRNAs.  Of 47 OTs, only 3 had mutations. Two of those sites were mutated in multiple mice, indicating fairly high rates at these particular OTs.   However, the mutated OTs all had only 2 or 1 mismatches, and the “high rate” OTs only had 1 mismatch near the 5’ end, distal to the seed region.   

Bottom line:  47 OTs screened, 3 mutated.

3. Li et al.  (Nat. Biotech. 2013).  Of 4 targets they used, only two had OTs with fewer than 4 mismatches or perfect seed matches, so they focused on those. 12 founders were screened. In a subset of pups also screened some more OTs that had perfect seed matches but were otherwise totally mismatched. 

Bottom line: more than 13 OTs screened, 0 mutated.

4.  Mashiko et al (Sci. Rep. 2013) were the first to publish on injecting plasmid DNAs into mouse zygotes for transient CRISPR expression.    Similar to Wang et al, they defined OTs as having a perfect match to the 12-13 bases adjacent to the PAM.  For two targets, they defined 7 and 4 OTs respectively; in 16 and 8 mutant pups made with either gRNA, they found one pup with a single OT mutation.

Bottom line:  11 OTs screened, 1 mutated.

5. Fujii et al (NAR 2013) targeted the Rosa26 locus, and reported that OT rates dropped as injected RNA concentrations were lowered.   They inspected 10 OTs with “3 or 4 mismatches” but each of these actually had a mismatch to the “N” of the PAM motif, which doesn’t affect targeting, so these were really “2” and “3” mismatched OTs.  No OT mutations were found for the true 3-mismatch OTs.  However they found mutations in all four 2-mismatch OTs,  particularly when injecting higher RNA concentrations .  They also examined 12 OTs for 2 targets in Hprt and found no OT mutations.

Bottom line:  22 OTs screened; 4 mutated but only in “2-mismatch” OTs.

6.   Wu et al (Cell Stem Cell 2013) targeted the Crygc gene and defined OTs as having no mismatches in a 14 base seed region of the target.  Of 10 OTs, 1 was mutated in 2 out of 12 pups.   

Bottom line:  10 OTs screened, 1 mutated.

7. Inui et al (Sci. Rep. 2014) examined about 10 OTs total for two targets, in Sox9 and Sf-1.

Bottom line:  ~10 OTs screened, 0 mutated.

8. Zhou et al (FEBS J. 2014) retargeted an EGFP cassette separately with two gRNAs.   OTs were defined using the MIT CRISPR design tool, and they analyzed a subset of these (15 OTs per target) by surveyor assay on the founder pups.  For one target, they detected mutations in 4 OTs, but not in any OTs for the second target.

Bottom line: 30 OTs screened, 4 mutated.

9. Mizuno et al (Mamm. Genome 2014) targeted the Tyr gene and screened 5 OTs in founders by sequencing.

Bottom line:  5 OTs screened, 0 mutated.

10.  Han et al (RNA Biol. 2014) used 4 targets and identified OTs with the MIT CRISPR design tool.  They screened 3 founders for the “top 5” potential OTs.

Bottom line:  20 OTs screened, 0 mutated.

  

Here’s a quasi-meta-analysis:

From these 10 studies, 5 (50%) were able to detect some degree of off-target mutation.

But from ~175 OT’s screened, mutations in only 13 (7%) were detected.  Several of these OTs had fewer than 3 mismatches to the target.

In conclusion, the consensus from many studies of CRISPR-mediated mouse engineering demonstrates that native Cas9 has a low rate of off-target effects in mouse zygotes.  Of course, targets should still be pre-screened when possible to avoid those that will have more potential off-targets, particularly those with fewer than 3 mismatches within the protospacer.   

Doug Mortlock 2014.

Bibliography

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2. Yang, H., H. Wang, C.S. Shivalila, A.W. Cheng, L. Shi, and R. Jaenisch, One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell, 2013. 154(6): p. 1370-9.

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4. Mashiko, D., Y. Fujihara, Y. Satouh, H. Miyata, A. Isotani, and M. Ikawa, Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci Rep, 2013. 3: p. 3355.

5. Fujii, W., K. Kawasaki, K. Sugiura, and K. Naito, Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucleic Acids Res, 2013. 41(20): p. e187.

6. Wu, Y., D. Liang, Y. Wang, M. Bai, W. Tang, S. Bao, Z. Yan, D. Li, and J. Li, Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell, 2013. 13(6): p. 659-62.

7. Inui, M., M. Miyado, M. Igarashi, M. Tamano, A. Kubo, S. Yamashita, H. Asahara, M. Fukami, and S. Takada, Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system. Sci Rep, 2014. 4: p. 5396.

8. Zhou, J., J. Wang, B. Shen, L. Chen, Y. Su, J. Yang, W. Zhang, X. Tian, and X. Huang, Dual sgRNAs facilitate CRISPR/Cas9-mediated mouse genome targeting. FEBS J, 2014. 281(7): p. 1717-25.

9. Mizuno, S., T.T. Dinh, K. Kato, S. Mizuno-Iijima, Y. Tanimoto, Y. Daitoku, Y. Hoshino, M. Ikawa, S. Takahashi, F. Sugiyama, and K. Yagami, Simple generation of albino C57BL/6J mice with G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system. Mamm Genome, 2014. 25(7-8): p. 327-34.

10. Han, J., J. Zhang, L. Chen, B. Shen, J. Zhou, B. Hu, Y. Du, P.H. Tate, X. Huang, and W. Zhang, Efficient in vivo deletion of a large imprinted lncRNA by CRISPR/Cas9. RNA Biol, 2014. 11(7): p. 829-35.