In vivo high-throughput profiling of CRISPR–Cpf1 activity

Abstract

CRISPR from Prevotella and Francisella 1 (Cpf1) is an effector endonuclease of the class 2 CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) gene editing system. We developed a method for evaluating Cpf1 activity, based on target sequence composition in mammalian cells, in a high-throughput manner. A library of >11,000 target sequence and guide RNA pairs was delivered into human cells using lentiviral vectors. Subsequent delivery of Cpf1 into this cell library induced insertions and deletions (indels) at the integrated synthetic target sequences, which allowed en masse evaluation of Cpf1 activity by using deep sequencing. With this approach, we determined protospacer-adjacent motif sequences of two Cpf1 nucleases, one from Acidaminococcus sp. BV3L6 (hereafter referred to as AsCpf1) and the other from Lachnospiraceae bacterium ND2006 (hereafter referred to as LbCpf1). We also defined target-sequence-dependent activity profiles of AsCpf1, which enabled the development of a web tool that predicts the indel frequencies for given target sequences (http://big.hanyang.ac.kr/cindel). Both the Cpf1 characterization profile and the in vivo high-throughput evaluation method will greatly facilitate Cpf1-based genome editing.

Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1

“…In contrast, LbCpf1/AsCpf1 modified the 4/3 CTTV, 3/4 TCTV, 4/3 TTCV, 2/2 CCTV, 3/2 TCCV and 1/0 CCCV sites, respectively (Figure 1D). These results revealed that LbCpf1 and AsCpf1 can edit the endogenous sites with the CTTV, TCTV or TTCV PAM, consistent with our in vitro cleavage data and a recent in vivo study (Kim et al, 2016c). Together, these results confirmed that, in addition to the canonical TTTV PAM, LbCpf1 and AsCpf1 can target CTTV, TCTV and TTCV as suboptimal non-canonical PAMs.…”

“…Similarly, in the AsCpf1 ternary complex, the 20-bp heteroduplex is accommodated within the REC lobe, in which Trp382, equivalent to Trp355 in LbCpf1, stacks with the C20:dG20 base pair, while AsCpf1 was crystallized with a 24-nt-guide-containing crRNA and its complementary target DNA (Yamano et al, 2016) (Figure 4D). Consistent with these structural findings, the crRNA-DNA base pairing in the PAM-distal region is dispensable for the Cpf1-mediated DNA cleavage (Zetsche et al, 2015; Kim et al, 2016b, 2016c; Kleinstiver et al, 2016). Together, these observations indicated that LbCpf1 and AsCpf1 recognize the 20-bp crRNA-target DNA heteroduplex.…”

“…LbCpf1 cleaved the plasmid target slightly more efficiently than AsCpf1 (Figure 1A). Previous studies indicated that, while LbCpf1 and AsCpf1 recognize TTTV (V is A, G or C) as the optimal PAM, they also recognize C-containing sequences, such as CTTV, TCTV and TTCV, as suboptimal PAMs (Kim et al, 2016c). To examine their preference for the fourth PAM nucleotide, we measured the cleavage activities of LbCpf1 and AsCpf1 toward four target sites, with either TTTA, TTTT, TTTG or TTTC as the potential PAM (Figure 1B).…”

“…More recently, the type-V Cpf1 (also known as Cas12a) effector nucleases from Acidaminococcus sp. BV3L6 (AsCpf1) and Lachnospiraceae bacterium ND2006 (LbCpf1) were identified and biochemically characterized (Zetsche et al, 2015), and they have been harnessed for genome editing in eukaryotic cells (Zetsche et al, 2015, 2016; Hur et al, 2016; Kim et al, 2016a, 2016b, 2016c; Kleinstiver et al, 2016; Tang et al, 2017). AsCpf1 and LbCpf1 share 34% sequence identity, while they lack similarity with Cas9 outside their RuvC domains.…”

“…Indeed, a recent study demonstrated that LbCpf1 and AsCpf1 can modify target sites with the non-canonical C-containing PAMs, such as CTTA, TCTA and TTCA, in mammalian cells, albeit with lower efficiencies than those with the canonical TTTV PAM (Kim et al, 2016c). However, the mechanism by which Cpf1 recognizes both the canonical and non-canonical PAMs has remained elusive.…”