Cas9

Alexandre Paix¹ and Geraldine Seydoux¹

¹HHMI and Johns Hopkins University School of Medicine, Deptartment of Molecular Biology and Genetics, Baltimore MD

Correspondence to: Alexandre Paix (apaix1@jhmi.edu)

Homology-directed repair (HDR) of double-strand breaks can be used to introduce precise edits in the genome, but is thought to be an inefficient process requiring long stretches of homology between donor and target sequences. We have found that HDR of cuts made by CRISPR/Cas9 is surprisingly efficient and requires only short homology arms, even for long, gene-sized edits (i.e., whole-gene deletions and GFP insertions).

Zhao et al. (2014) reported earlier this year that single-strand DNA oligos (ssODNs) can be used to introduce base-size edits near Cas9 sites without selection. Inspired by that report, we tested whether ssODNs could also be used to introduce small insertions, as shown for TALEN sites by Lo et al., (2013). We designed ssODNs to contain a restriction site (7 bases) or protein tags (18 to 66 bases: Tetra-Cys,V5, Myc, OLLAS, and FLAG) flanked by short homology arms (50-60 bases) and screened for edits in the first generation after injection (F1), using PCR and restriction digestion as in Zhao et al., (2014). We identified edits at frequencies ranging from 0.9 to 7% of F1s. Edit frequency was affected more by sgRNA efficiency than by edit size. We also found that ssODNs that bridge two sgRNA cut sites spaced far apart can be used to generate a precise, whole-gene deletion (1.7 kb, 3.8% of F1s). (Imprecise gene-size deletions can also be generated using two spaced sgRNAs and no repair template).

Emboldened by these results, we tried the same approach to insert GFP. We used PCR fragments made by amplifying GFP (864 bases) with primers containing ~60 bases flanking the Cas9/sgRNA site, inserting the GFP right in the sgRNA site or 27 bases away. We obtained 4% and 0.9% GFP-positive F1s overall, with 15% of injected mothers giving 20-48% edited F1s on the second day after injection (“jackpot broods”). The edits can be identified directly among the F1s by PCR or, most efficiently, by direct inspection for GFP expression. In this way, it is possible to go from injection to beautiful GFP pattern in just four days!

Based on these results, we developed a systematic protocol to mutate, delete, or tag any gene using ssODNs or PCR fragments. Using this protocol, the lab has already modified 13 genes. We are currently testing whether our approach could be streamlined even further using Co-CRISPR schemes to identify jackpot broods (Kim et al., 2014; Zhang and Glotzer, 2014; Arribere et al., 2014).

Update: Paper (including protocol) is now in press at GENETICS (http://www.genetics.org/content/early/2014/09/30/genetics.114.170423).

References

Arribere JA, Bell RT, Fu BX, Artiles KL, Hartman PS, and Fire AZ. (2014). Efficient marker-free recovery of custom genetic modifications with CRISPR/Cas9 in Caenorhabditis elegans. Genetics pii: genetics. 114. 169730. doi: 10.1534/genetics.114.169730

Kim H, Ishidate T, Ghanta KS, Seth M, Conte D Jr, Shirayama M, and Mello CC. (2014). A Co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans.

Lo TW, Pickle CS, Lin S, Ralston EJ, Gurling M, Schartner CM, Bian Q, Doudna JA, and Meyer BJ. (2013). Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs and CRISPR/Cas9 to engineer insertions and deletions. Genetics 195, 331-348.

Paix A, Wang Y, Smith H, Lee CY, Calidas D, Lu T, Smith J, Schmidt H, Krause M, and Seydoux G. (2014). Scalable and versatile genome editing using linear DNAs with micro-homology to Cas9 sites in Caenorhabditis elegans. Genetics (2014). Sep 23. pii: genetics.114.170423. [Epub ahead of print]

Zhang, D. and Glotzer, M. (2014). Efficient site-specific editing of the C. elegans genome. bioRxiv doi: http://dx.doi.org/10.1101/007344 BioRxiv

Zhao P, Zhang Z, Ke H, Yue Y, and Xue D. (2014). Oligonucleotide-based targeted gene editing in C. elegans via the CRISPR/Cas9 system. Cell Res. 24, 247-250.

Wadim Kapulkin1 and Mihail Sarov1

1Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

Correspondence to: Wadim Kapulkin (kapulkin@mpi-cbg.de)

Inspired by work from the Calarco lab (Friedland et al., 2013) concerning CRISPR/CAS9, I have been investigating if unc-22 could be utilized as a convenient marker for CRISPR/CAS9 system activity. A single sgRNA guide (GGAGAAGGAGGCGGTGCTGG) was designed to target the unc-22 locus (termed unc-22-sgRNA-1000, since it was the 1000^th sgRNA of those predicted to lay within the unc-22 gene). Since Friedland and colleagues demonstrated simultaneous CRISPR activity of two independent sgRNAs on two different loci, I assumed that it would be useful to evaluate/discriminate between transformed animals by selecting only those that engage the proper CAS9 activity. Below, I describe three experiments that show that the above unc-22-sgRNA-1000 worked consistently and efficiently in my hands.

Experiment 1

In a preliminary attempt, I injected unpurified PCR product (synthesized by PCR stitching, resulting in pU6:unc-22-sg1000) diluted with an injection mix containing ~200ng/µl of peft-3::CAS9 encoding plasmid and fluorescent markers, into 12 him-6(e1104) animals (Hodgkin et al., 1979). 9 P0 plates segregated some number of marker positive F1’s. Among 60 singled F1’s, I recovered one plate with apparent F2 twitchers. Upon sequencing, I confirmed 2 lesions in the guide-specified region [a (-9bp) deletion resulting in weak twitcher phenotype and a (-42;+2 indel) resulting in a strong twitcher phenotype] transmitted to F3.

Experiment 2

In the second experiment, the above pU6:unc-22-sg1000 was injected as a plasmid (~25 ng/µl in the same injection mix as in Experiment 1) into 24 him-6(e1104) animals. Among the first 60 marker positive F1’s, I isolated F1 Twitcher. This phenotypicaly affected F1 unc-22(-) animal enthusiastically segregated a strong twitching phenotype in the F2. Sequencing confirmed 2 lesions in the unc-22-sgRNA-1000 specified region [(-61bp) and (-105bp) deletions] transmitted to F3.

Experiment 3

In this attempt I injected the mix used in Experiment 2 into animals of a more complex genotype [smg-1(cc546ts); dvIs27] (Link et al., 2003). A temperature sensitive smg-1(cc546ts) mutation was developed by the Fire lab as a means to engineer conditional transgene expression (see WBG 14 (5) February, 1997, Getz et al., Fire lab vector kit 97, and source vectors www.addgene.org/static/cms/files/Vec97.pdf for detailed description). Surprisingly, among the first 60 marker positive F1’s, three animals (of two independently injected P0) segregated spectacularly spastic F2 twitchers. Sequencing of the progeny of the above F2 twitchers confirmed at least 8 different lesions in the unc-22-sgRNA-1000 specified region [(-61bp), (-42bp), (-32bp), (-15bp), (-12bp), (-9bp) deletions and two indels (-51;+7bp) and (-26;+7bp)]. At least three other F1’s (all siblings of the above strong F1) segregated weaker twitchers that contained two additional small frame restituting deletions [(-9bp) and (-15bp)]. Interestingly, amongst the singled F2 progeny, we recovered two sibling twitcher lines with a confirmed (-61bp) deletion growing considerably slower than the other lines. Both lines contained homozygous insertions of the extrachromosomal array, presumably containing peft-3::CAS9 and pU6:unc-22-sgRNA-1000 plasmids.

From the above experiments I conclude that both him-6(e1104) and smg-1(cc546ts); dvIs27 appear permissive for the unc-22–-sgRNA-1000 dependent CAS9 effects on unc-22. Due to some smg-1(ts) properties or the presence of dvIs27 (possibly aggravating the twitches), smg-1(cc546ts); dvIs27 might be somehow favorable in recovering heritable unc-22 phenotypes (however both, above explanations are not mutually exclusive and both remain speculative). On the other hand, him-6(e1104) might perhaps sometimes lead to early identification of heritable unc-22 phenotypes in the F1.

The most surprising observation was that some identified deletions (-61bp), (-15bp), and (-9bp) were confirmed in the F2’s from independently injected parental animals. Strikingly, an identical (-61bp) deletion was identified in the experiments conducted in him(-) and smg(-) backgrounds. Of the above observation, I conclude that in the independent experiments the most prevalent lesion [i.e., the (-61bp) deletion] apparently reoccurs. This may imply that the initial CAS9 cleavage specified by unc-22-sgRNA-1000 operates under some genomic constraint (e.g., due to the local chromatin structure or other intrinsic properties of the specified region), and/or perhaps the repair mechanism is biased.

References

Friedland AE, Tzur YB, Esvelt KM, Colaiacovo, MP, Church GM, and Calarco, JA. (2013). Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat. Methods 10, 741-743.

Hodgkin J, Horvitz HR, and Brenner S. (1979). Nondisjunction mutants of the nematode Caenorhabditis elegans. Genetics 91, 67-94.

Link CD, Taft A, Kapulkin WJ, Duke K, Kim S, Fei Q, Wood DE, and Sahagan BG. (2003). Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer’s disease model. Neurobiol. Aging 24, 397-413.

The WBG

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A fast, marker-free method to mutate, tag, or delete any gene using linear DNAs with short homology arms

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CRISPER’ed twitchers

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