What is the minimum required length of homology arms for CRISPR-mediated genome editing?

I don't think this answer is well understood yet - or at least, not published.   We know from the Jaenisch lab papers that single-stranded oligos having 60 nt of homology on either side of the target are functional to direct homology-directed repair (HDR).   It's not clear that double-stranded molecules would be better; in fact, they might be targets for CRISPR cleavage too, depending on the exact design of the target & editing design.  (Single-stranded DNAs are presumably not recognized by the Cas9 as targets for cleavage, especially if they correspond to the same strand of the protospacer as they can't base pair with the guide RNA).  

So, ssDNA oligos are fine for inserting loxP sites or peptide tags as we can order "ultramers" of up to 200 nt+ from IDT, etc. but this won't be practical for inserting longer cassettes like GFP reporters.   Here I think the jury is still out on the length of homology arms needed.    ~ 1 kb arms seem to be the ballpark for published accounts of reporter insertion, via CRISPR.   Bigger is probably better, but what's minimally necessary for efficiency?

Improving the CRISPR guide RNA / Live imaging of tagged genomic loci with CRISPR/Cas

Back in October, Chen et al. reported in Cell the use of an optimized CRISPR guide RNA and endonuclease-deficient/EGFP-fused Cas9 to mark specific loci in live cell nuclei.   Their first experiments were to mark telomeric sequences, and in so doing they discovered that the guide RNA was a rate-limiting factor for efficient targeting.   They then made 2 improvements to the same "long-form", chimeric guide RNA that's been widely used; these changes to the guide RNA are as follows:

1.  U to A change at position #5 downstream of the DNA-binding portion. Purpose:  Disrupts a 4-U stretch that may cause premature termination of guide RNA transcripts.

2.  Extension of the proximal stem-loop by 5 base-pairs.  Purpose: probably stabilizes interaction with Cas9.

This enhanced specific target labeling even while EGFP-Cas9 levels were reduced, which in turn lowered nonspecific background fluorescence.   It's likely that these guide RNA modifications will generally increase Cas9 targeting efficiency in other context.  Thanks Ian for bringing this to my attention.

Interesting example of CRISPR mouse knockout strategies

Many traditional mouse knockouts have generated results that were not straightforward to interpret, or even controversial.   CRISPR promises to allow more replication experiments, or varying targeted designs, to sort out some of these issues as discussed in the Prp/Prnp story:

http://www.cureffi.org/2014/03/09/how-to-and-how-not-to-knock-out-prnp/

Vanderbilt CRISPR interest group April 4 noon MRB3

Dr. Ian Macara and I are initiating a Vanderbilt CRISPR interest group.  The first meeting is Friday April 4 at noon in 3131 MRB3.   "If you have begun using CRISPR for any project we would be interested in hearing a brief, 3 – 5 minute presentation of successes, failures, problems, etc."   It's intended to be an open discussion to exchange ideas and technical tips.   Feel free to attend and to let other people know who want to attend.

PX330 update

Preliminary indications are that we are having success with using the PX330 plasmid to mutagenize mouse embryos following pronuclear injection.

UC Berkeley launches a CRISPR Center

A $10 million gift has been given to Berkeley to start a Center for Genomic Engineering with Jennifer Doudna as its leader.  Doduna was a co-senior author on the seminal Jinek et al 2012 paper in Science describing CRIPSR/Cas9 functionality.

Jackson Lab hosting a webinar about CRISPR/Cas for mice on March 27

(Reposted from a JAX email.)

Don’t miss the special JAX Webinar^TM: CRISPR/Cas Mediated Genome Engineering in Mice

Date: Thursday March 27, 2014

Time: 10am PT US/ 11am MT US / 12pm CT US / 1pm ET US

Please follow this link to register now for this complementary webinar:

March 27 Webinar (CRISPR/Cas)

  

Presenter:

Haoyi Wang, Ph.D., Adjunct Faculty, The Jackson Laboratory

Moderators:

Wenning Qin, Ph.D., Associate Director, Genetic Engineering Technologies, The Jackson Laboratory

Kathy Snow, Ph.D., Manager, Technical Information Services, The Jackson Laboratory

Webinar Description

Targeting multiple genes in one generation? Systematically modulating individual components of a system in a few years rather than decades? The promises of precise, combinatorial, efficient methods for genomic engineering are exciting. The CRISPR/Cas technology applied to mouse genetic engineering could quickly advance scientific understanding of disease mechanisms by allowing researchers to ask complex questions and find answers much faster than with traditional gene targeting approaches.

During this webinar, Dr. Haoyi Wang, one of the developers of this revolutionary technology will share how CRISPR/Cas has been used successfully in mice to generate endogenous knock-in alleles, conditional (“floxed”) mutations, as well as multiple mutations in a single generation. Join us for this presentation as we discuss the following topics:

·         How CRISPR/Cas9 works to modify host genomes

·         How this technology compares to similar technologies

·         Ways in which CRISPR/Cas9 has been used to make targeted mutations and insertions

·         Technical challenges and practical considerations

Register today and don’t miss out on this opportunity to learn from a pioneer in this new revolutionary technology.

________________________________
Shannon Byers, MS, MBA
Reproductive Sciences R&D
The Jackson Laboratory (box 26)
600 Main Street
Bar Harbor ME 04609
1.207.288.6726

3rd generation CRISPR/Cas9 vectors from Zhang lab

The Zhang lab has made available 2 new plasmids that are like PX330 but with T2A-puromycin or T2A-eGFP linked to Cas9:  PX459 and PX458.

Constraints on CRISPR target choice

This is my graphic summary of "best practices" for CRISPR target choice, as of today anyway.  Here's additional thoughts:

• The protospacer can apparently be truncated from 20 bases to as few as 17 bases with essentially no loss in cleavage efficiency; however, 18- or 17-base protospacers had significantly reduced levels of off-target cleavage (see Fu et al Nat Biotech 2014).  To me, 18 bases is the sweet spot of length that allows efficiency and maximizes specificity while still minimizing the risk of highly similar off-targets being present in animal genomes.  17 bases is still good for avoiding identical and even single-base mismatches in genomes.  However from the Fu et al paper some of the 17 base truncations did have a noticeable drop in efficiency (although many worked as well as 20 bases), whereas 18 base tru-gRNAs all seemed to work well.

• The G at the 5' end facilitates transcriptional initiation by the U6 promoter, e.g. in the PX330 vector and its derivatives.   It's not required by CRISPR/Cas9 per se.   It seems to be possible to simply add a G onto the 5' end of any N20 protospacer, according to Mashiko et al (Dev Growth Differ 2013): "If the first nucleotide was not G, we added an extra G at the 5' end, as the U6 promoter prefers a G for transcriptional initiation."   However, it's not clear if the protospacer can be truncated and an extra G can be added at the 5' end - these haven't been tested.  I think the answer is probably yes, because Fu et al showed that the first 2 bases of 3 different N20 guide RNA protospacers can be altered with minimal or no drop in efficiency.

• A T-stretch of 5 or more will terminate RNAPol III transcription, so avoid those if the U6 promoter is being used.

• Base preferences in the protospacer: None seem to be prohibited.  In the Wang et al Science paper (2014 issue 6166), they showed that U bases in the last 4 protospacer positions are apparently measurably detrimental to sgRNA loading onto Cas9, especially at positions -1, -2, and -4, when data from high-throughout target screening was analyzed.   However it's pretty clear that many targets cut well if they have a T or two there, and some that have poor efficiency have no Ts there.