Paper: In vivo interrogation of gene function in the mammalian brain using #CRISPR-Cas9.

Swiech et al. used AAV vectors to mutate target genes in adult mouse brains.     This paper is notable for the careful measurement of on-target mutation efficiencies in vivo, including from single cell nuclei, which strongly indicated that about ~65% of transduced brain cells acquired mutations in both alleles of their initial target gene (Mecp2).  Off-target effects were apparently low (0-1.6 % rates of mutation at the "top predicted off-target" for each of 3 Dnmt family genes, measured in GFP+ cells).   With their AAV vectors, one vector supplies Cas9 production and the other expresses the guide RNA and also GFP; thus fluorescence indicates transduction.  They also introduce an AAV vector that coexpresses up to 3 guide RNAs + GFP for multiplexed targeting.

In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9

• Lukasz Swiech,
• Matthias Heidenreich,
• Abhishek Banerjee,
• Naomi Habib,
• Yinqing Li,
• John Trombetta,
• Mriganka Sur
• & Feng Zhang.

Nature Biotechnology.   19 October 2014

Poly Peak Parser can be useful for identifying new #CRISPR indels in F1s using PCR + Sanger chromatogram data.

Thank you to my colleague Max for pointing this out to me.  If you are doing CRISPR on mice, fish or whatever creatures you are working one, and you are direct-Sanger-sequencing PCR products from #CRISPR animals, you know that animals with more than one type of allele will generate confusing, overlapping sequence traces usual extending past the CRISPR cut site.  This is because CRISPR (or TALENS and ZFNs for that matter) usually generates indel mutations.   Although the sanger data is fine to confirm something got altered by CRISPR, the overlapping peaks make it hard to identify exactly what the indel is.     Poly Peak Parser is an easy to use web interface for pulling the alternate allele (e.g. the newly generated indel) out of an .abi or .scf file that has double peaks due to indel heterozygosity.  It is designed to be used on PCR Sanger data from F1 heterozygote animals.  

Poly peak parser: Method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products.Hill JT, Demarest BL, Bisgrove BW, Su YC, Smith M, Yost HJ.Dev Dyn. 2014 Aug 27. doi: 10.1002/dvdy.24183. [Epub ahead of print]         

Web tool:   http://spark.rstudio.com/yostlab/PolyPeakParser/

The catch is that you have to supply the reference allele sequence (such as wild type), and it really only works well if there are 2 and only 2 alleles embedded in the sanger data, one of which is the reference sequence.  Then it will extract the alternate allele from the double peak data.    I tried it out using sanger data from a mouse that was confirmed to be a heterozygote for wild type allele + a new, short deletion; Poly Peak Parser quickly returned the alternate allele confirming the 1 bp deletion.   

In practice, however, founder animals from CRISPR injections are usually not simply heterozygous for wild type and a new indel allele.  They usually have at least two mutated alleles and sometimes more, if they are mosaic.  As the authors of this tool state in their paper, Poly Peak Parser is really designed for analyzing F1 animals.   So here is a suggested workflow:

1.  If you have sanger files from PCRs of founder animals and they clearly have double peaks, try inputting the .scf along with a wild type reference sequence into Poly Peak Parser and see if it returns an alternate allele that looks like it mostly has unambiguous base calls.   

2. If the "alternate allele" has lots of ambiguous bases, the animal may be mosaic, or simply has two new but distinct mutations.

Either way, breed founder animals to wild type to get F1s and the data will be much more clear.

More protocols for mouse mutagenesis with #CRISPR: newly published in CPHG.

Harms et al have created this detailed set of protocols for conducting CRISPR/Cas9 mutagenesis and editing in mouse embryos.  Included are more guidelines and instructions for choosing targets, then on to ligation protocols for cloning protospacers in sgRNA expression vectors, in vitro RNA synthesis, pronuclear injection, and follow-up screening/genotyping of founder animals, as well as HDR donor design and considerations.  Also a timeline schematic.   


Mouse Genome Editing Using the CRISPR/Cas System.Harms DW, Quadros RM, Seruggia D, Ohtsuka M, Takahashi G, Montoliu L, Gurumurthy CB.Curr Protoc Hum Genet. 2014 Oct 1;83:15.7.1-15.7.27. doi: 10.1002/0471142905.hg1507s83.

A review about #CRISPR off-target effects with links to 10 online off-target identification tools.

Gene editing: how to stay on-target with CRISPR.  Vivien Marx.  Nat Methods. 2014 Sep 29;11(10):1021-6. doi: 10.1038/nmeth.3108.

In addition, let me reiterate some rules of thumb about minimizing off-target effects:

1.  3 or more mismatches in the 20-base protospacer are highly likely to prevent off-target cleavage.

2.  Or, consider using truncated protospacers of 17 or 18 bases.  The catch is you will probably have to pick a protospacer with a G as the first base to allow guide RNA transcriptional initiation.

3. I'm not sure if paired nickases are the best way to go yet.  A better way to think of it is that it may depend on the specific goal of your CRISPR experiment.      

Outstanding #CRISPR review: "A mouse geneticist's practical guide to CRISPR applications".

 Far above Cayuga's waters - my alma mater! - there are some excellent scientists using CRISPR to engineer mutations in mice.   This paper is from John Schimenti's lab at Cornell and I believe it contains the most thorough review of  mouse CRISPR engineering to date.   

A Mouse Geneticist's Practical Guide to CRISPR Applications.  Singh P, Schimenti JC, Bolcun-Filas E.  Genetics. 2014 Sep 29. pii: genetics.114.169771. [Epub ahead of print]

PMID:25271304

More than merely a review article, it has some additional new data from this group.  They tested whether inhibition of the NHEJ pathway could enhance efficiency of CRISPR-mediated homology-directed repair, since these pathways compete following CRISPR cleavage; this was based on similar experiments done in Drosophila using ZFNs to do HDR.   The answer seems to be yes, it helps in mouse embryos as well.  The compound they used was SCR7, an inhibitor of ligase IV (a key player in NHEJ).   Crucially, it seems that SCR7 can be applied directly to mouse embryo culture media with minimal toxicity.   Go to Table 2 for the result suggesting a shift in balance from predominantly NHEJ alleles toward more HDR alleles.     

Quoting from a new #CRISPR paper: "..requirements for Cas9 DNA binding are different from those for catalytic activity"

The paper is :  Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage. Cencic R, Miura H, Malina A, Robert F, Ethier S, Schmeing TM, Dostie J, Pelletier J.  PLoS One. 2014 Oct 2;9(10):e109213. doi: 10.1371/journal.pone.0109213. eCollection 2014.  

Two previous papers (Wu et al and Kuscu et al) showed that Cas9/guide RNA complexes are normally resident on many genomic sites, the vast majority of which do not get cleaved by Cas9.  Thus, binding does not automatically lead to cleavage - far from it.   The data in this paper clearly indicates the requirement of the PAM-proximal "seed" portion of the protospacer for initial Cas9/sgRNA binding, while the distal protospacer does not need to match to permit binding.  However, the additional pairing of the more distal portion of the protospacer is required for target cleavage.  Thus binding and cleavage are distinct steps.

One important implication of these studies is in regard to using cleavage-deficient Cas9, e.g. with both DNAse domains mutated, to physically localize fusion complexes (GFP, transactivation domains, etc) to genomic targets for reasons other than genomic editing.   Examples include fluorescent labeling of telomeres and upregulation of specific target genes. (Cheng et al and Perez-Pinera et al).

The results from this and the previous papers now clearly suggests that for CRISPR-Cas9 applications that only require binding to the target, not cleavage, off-target effects may be more widespread.   Whether that will be an serious problem, and maybe it won't be, will probably be very dependent on the application in question.  

In any event these papers provide data that may point the way to further increasing CRISPR/Cas9 specificity, in a variety of different applications and settings.