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Small Molecules boost Cpf1-mediated Genome Editing in Pluripotent Stem Cells

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Review of “Small molecules promote CRISPR-Cpf1-mediated genome editing in human pluripotent stem cells” from Nature Communications by Stuart P. Atkinson

While CRISPR-Cas9 genome editing in human pluripotent stem cells (hPSCs) has stolen the headlines of recent times, another CRISPR system employing Cpf1 also holds great potential [1]. Smaller size and simpler characteristics, when compared to Cas9, makes Cpf1 more suitable for multiplexed genome editing with low off-target activity [2-4]; however, the suitability of CRISPR-Cpf1 to genome editing in hPSCs remains mostly unexplored.

To this end, a new study from the laboratory of Saiyong Zhu (Zhejiang University, Hangzhou, China) now demonstrate the efficiency of CRISPR-Cpf1-mediated genome editing in hPSCs and describe their search for small molecules that can enhance this process even further [5].

Ma et al. employed U6 promoter-driven CRISPR-RNA expression plasmids to ensure the specific CRISPR-Cpf1-mediated editing (knockout) of two genes of interest - ALKBH1, a tRNA demethylase, and CLEC16A, involved in diabetes - creating systems that displayed efficient indel rates in both hESCs and hiPSCs with low off-target activities. To then test for chemical compounds that can improve CRISPR-Cpf1 gene editing in hPSCs, the authors studied the knock-in of a puromycin resistance cassette into the OCT4 target locus. Encouragingly, two compounds (VE-822, a specific inhibitor of Ataxia Telangiectasia mutated and Rad3-related kinase (ATR), and AZD-7762, a specific inhibitor of checkpoint kinase CHEK1) significantly enhanced the initial low levels of CRISPR-Cpf1 knock-in activity. Furthermore, both compounds enhanced point mutation editing using a short single-stranded oligodeoxynucleotide (ssODN) template, suggesting that CRISPR-Cpf1 and small molecules can combine to promote efficient genome editing in hPSCs.

The authors hope that the discovery of VE-822 and AZD-7762 will promote the widespread application of CRISPR-Cpf1 as a simple and efficient strategy for precise genome engineering of hPSCs.

To keep up with all the following research papers on CRISPR-Cpf1 gene editing in hPSCs and all the related applications, stay tuned to the Stem Cells Portal.

References

  1. Zetsche B, Gootenberg Jonathan S, Abudayyeh Omar O, et al., Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 2015;163:759-771.
  2. Kim D, Kim J, Hur JK, et al., Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nature Biotechnology 2016;34:863.
  3. Kleinstiver BP, Tsai SQ, Prew MS, et al., Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nature Biotechnology 2016;34:869.
  4. Zetsche B, Heidenreich M, Mohanraju P, et al., Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array. Nature Biotechnology 2016;35:31.
  5. Ma X, Chen X, Jin Y, et al., Small molecules promote CRISPR-Cpf1-mediated genome editing in human pluripotent stem cells. Nature Communications 2018;9:1303.