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

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.


  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.