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However, their dependence on a 5\u2032-TTTV-3\u2032 protospacer adjacent motif (PAM) next to DNA target sequences restricts Cas12a\u2019s gene targeting capability to only \u223c1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a\u00a0with expanded PAM recognition. The resulting Cas12a variants use a range of noncanonical PAMs while retaining recognition of the canonical 5\u2032-TTTV-3\u2032 PAM. In particular, biochemical\u00a0and cell-based assays show that the variant Flex-Cas12a utilizes 5\u2032-NYHV-3\u2032 PAMs that expand DNA recognition sites to \u223c25% of the human genome. 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