Friday, January 31, 2014

Genetic engineering of monkeys using CRISPR

For earlier CRISPR posts, see here. Off-target mutations seem to be under control in this study. Technology Review.
Generation of Gene-Modified Cynomolgus Monkey via Cas9/RNA-Mediated Gene Targeting in One-Cell Embryos (DOI:10.1016/j.cell.2014.01.027)

Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, we successfully achieve precise gene targeting in cynomolgus monkeys. We also show that this system enables simultaneous disruption of two target genes (Ppar-γ and Rag1) in one step, and no off-target mutagenesis was detected by comprehensive analysis. Thus, coinjection of one-cell-stage embryos with Cas9 mRNA and sgRNAs is an efficient and reliable approach for gene-modified cynomolgus monkey generation.

In other exciting news: Bidirectional developmental potential in reprogrammed cells with acquired pluripotency (DOI:10.1038/nature12969): stimulus-triggered acquisition of pluripotency (STAP).


DK said...

no off-target mutagenesis was detected by comprehensive analysis

I was about to write "wow, amazing, how is it even possible and yes, I was dead wrong on this" but then opened up the actual paper. And I am afraid that this is the case of a fundamental lack of basic understanding how enzymes (and DNA-modifying enzymes in particular) work. From the methods:

All potential off-target sites with homology to the 23 bp sequence (sgRNA+PAM) were retrieved by a base-by-base scan ... potential off-target sites with less than three mismatches in the seed sequence (1 to 7 base) were selected to PCR amplification

Emphatically, that is *not* a proof that no off-target incorporation has occured!!! Vast majority of off-target modifications are expected to be ~random, in stretches of DNA having no similarity to the targeted sequence(s). That's because with low probability Cas9 will cut anywhere - like any nuclease. (The term "homology" is also used incorrectly above - but that's a common error). The *only* way to see how many non-specific mutations one round of CRISPR/Cas9 introduces is to do a FULL sequencing of the progeny and compare it to the parents and non-modified siblings. Back of the envelop calculations say that there should be ~10 non-specific events per each specific.

To be sure, this is an amazing and highly technical work that is awe-inspiring, and yet it's not what it says it is.

will said...

Awesome. Myopia may be wiped entirely from the gene pool.

BobSykes said...

While I share DK's reservations, I hope the authors and their institution have installed proper security for both the laboratory and the researchers and staff. This sort of thing will incense animal rights activists and environmentalists, and they will at some point launch a violent attack on the facilities and people involved.

steve hsu said...

What do you think of this paper?

"Here, we show that off-target effects of RGENs can be reduced below the detection limits of deep sequencing by choosing unique target sequences in the genome and modifying both guide RNA and Cas9. ... Our results highlight the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to avoid or reduce RGEN-induced off-target mutations."

It seems like the matching of guide RNA to target region is the crucial thing. Is there really uncontrollable random cutting?

steve hsu said...

This lab is, I believe, in Kunming. They don't have the same issues we have here ...

DK said...

"exome sequencing analysis showed that no off-target mutations were induced"

So, they are looking at only 5% of the genome... I.e., if the ratio is 1:10, they can't detect it. They also excluded excess point mutations and have done it completely arbitrarily: "Because engineered nucleases rarely induce substitutions via error-prone NHEJ". The reference is to analysis of complex mutations by very different nucleases. Right. Because all nucleases are the same. And the "rarely" is not defined but definitely not below 10^(-6).

Is there really uncontrollable random cutting

There has to be. Only a matter at what frequency. The rule of thumb in biology is that specificity ratio almost never exceeds 10^6. Polymerases make errors, transcription factors start off random sequences, enzymes modify "wrong" substrates, etc, etc - all this happens, but very rarely. The best characterized "specific" nucleases, type II restriction enzymes, will occasionally cleave off-target sequences - they have to because binding and catalytic parts are separate and the cleavage specificity comes bringing catalytic center into proximity of the substrate. Specificity of recognition is not, and cannot be unlimited - non-specific binding is a fact of life (with low affinity, everything interacts with everything).

steve hsu said...

If they see no off-targets in 5% (exomes) of the genome then (assuming uniform probability of random cutting), there might be ~ 10 at most in the rest of the genome -- it depends on how many individuals they did the exomes for. If you did, e.g., 20 individuals and found no off-targets on their exomes then you would be pretty confident that off-target probability is really small. IIUC, in the paper linked above they claim no off-targets in 4 clonal populations, which suggests less than 10 in the whole genome of an individual. I seem to recall other papers, perhaps mouse or human stem cell, that also did deep sequencing looking for off-targets.

For therapeutic purposes, you'd probably be willing to tolerate ~10 random mutations in exchange for eliminating something very bad.

Endre Bakken Stovner said...

You wouldn't want that. IQ and nearsightedness are genetically overlapping phenomena. See Cohn, Cohn and Jensen: IIRC they tested within-famliy correlation between IQ and myopia in sixty sibling pairs and found highly significant results.

There are more recent reviews, but pubmed wasn't good to me on this Sunday morning.

There are intrinsic relationships between asthma and high IQ too.

Anonymous_IV said...

Are these relations truly genetic, or just cultural artifacts? A folk explanation in both cases is that a kid with asthma or poor eyesight is less likely to be playing sports or scouting outdoors etc., and later less active on the dating scene, and will thus have more time to read, study, and think, and will tend to socialize with other kids of similar interests.

Endre Bakken Stovner said...

There can certainly be synergistic effects, but there are very good reasons for believing it to be partly explained by genetic overlap.

A from the top of my head rebuttal would be that there are many other reasons a person might be less socially active or play much, for example being born into or having to use a wheelchair from a young age. If the folk explanation for the overlap between myopia and intelligence were correct you should also expect people using wheelchairs from a young age to be more intelligent.

Another reason to think that there is partial genetic overlap is that the myopic are rather more intelligent than average, something like half a sigma in several studies done in different countries by different researchers, and that there is slim evidence of your IQ increasing that much merely by studying.

Furthermore, in the Cohen study linked to above (article beyond paywall here: you'll see that there was no difference in the time spent studying between the gifted and less gifted siblings, but the gifted were still more myopic.

A more recent study of ~1000 children from Singapore estimated that IQ was a greater risk factor for myopia than reading: "After controlling for age, gender, school, parental myopia, father’s education, and books read per week, myopia (spherical equivalent [SE]) of at least −0.5 D was associated with high nonverbal IQ"

will said...

How is it then that many geniuses and many High IQ people manage to not have the refractive error of myopia? If that is so. Then Myopia can be eliminated without impacting IQ.

will said...

Therefore myopia is incidentally associated with particular genetic makeup that cause IQ but not necessarily so.

Endre Bakken Stovner said...

If the overlap wasn't perfect, you could still have gifted people without visual impairment.

This is where a bivariate GCTA analyis would be helpful: (Finding out for sure is more fun than discussing on the internet.)

Michael Bacon said...

You say that there is slim evidence of your IQ increasing that much merely by studying, but what about high IQ folks just reading more and causing eye problems? Just curious. Thanks.

will said...

Thanks for the link. But back to my original point, myopia therefore can be eliminated and IQ not negatively impacted provided the correct genetic makeup put in its place. Mental capabilities need not come at the expense of physical ailment.

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